Microfluidic Cell Trap and Assay Apparatus for High-Throughput Analysis

ABSTRACT

Microfluidic devices are provided for trapping, isolating, and processing single cells. The microfluidic devices include a cell capture chamber having a cell funnel positioned within the cell capture chamber to direct a cell passing through the cell capture chamber towards one or more a cell traps positioned downstream of the funnel to receive a cell flowing. The devices may further include auxiliary chambers integrated with the cell capture chamber for subsequent processing and assaying of the contents of a captured cell. Methods for cell capture and preparation are also provided that include flowing cells through a chamber, funneling the cells towards a cell trap, capturing a predefined number of the cells within the trap, interrupting the flow of cells, flowing a wash solution through the chamber to remove contaminants from the chamber, and sealing the predefined number of cells in the chamber.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention relates to microfluidic devices. In particular, theinvention relates to microfluidic devices and their uses and methods forassaying cells.

2. Description of Related Art

Single cells represent a fundamental biological unit. However, the vastmajority of biological knowledge has emerged as a consequence ofstudying cell populations and not individual cells. Inevitably, thereare fundamental and applied questions, such as those relating totranscriptional control of stem cell differentiation, intrinsic noise ingene expression, and the origins of disease, that may only be addressedat the single cell level. For example, single cell analysis allows forthe direct measurement of gene expression kinetics, or for theunambiguous identification of co-regulated genes, even in the presenceof de-synchronization and heterogeneity that could obscurepopulation-averaged measurements. Similarly, single cell methods arevital in stem cell research and cancer biology, where isolatedpopulations of primary cells are heterogeneous due to limitations inpurification protocols, and it is often a minority cell population thatis the most relevant. High-throughput single cell measurementtechnologies are therefore of interest and have broad applications inclinical and research settings.

Existing methods for measuring transcript levels in single cells includeRT-qPCR (1), single molecule counting using digital PCR (2) orhybridization probes (3, 4), and next generation sequencing (5). Ofthese, single cell RT-qPCR provides combined advantages of sensitivity,specificity, and dynamic range, but is limited by low throughput, highreagent cost, and difficulties in accurately measuring low abundancetranscripts (6).

Microfluidic devices employing active valving to position and isolatecells have allowed for the isolation and genome amplification ofindividual microbial cells (32). Unfortunately, such devices do notallow for high throughput analysis due to the manual effort involved inoperating the valving mechanisms. Moreover, the device does not allowisolated cells to be washed from the supernatant prior to treatment oranalysis. This in turn allows for contamination events, and furtherlimits the downstream applications of the device.

Accordingly, a goal of microfluidics research has been the developmentof integrated technology for scalable analysis of transcription insingle cells. Microfluidic systems provide numerous advantages forsingle cell analysis: economies of scale, parallelization andautomation, and increased sensitivity and precision that comes fromsmall volume reactions. Considerable effort over the last decade hasbeen directed towards developing integrated and scalable single cellgenetic analysis on chip (7, 8). Thus, many of the basic functionalitiesfor microfluidic single cell gene expression analysis have beendemonstrated in isolation, including cell manipulation and trapping (9,10), RNA purification and cDNA synthesis (11-13), and microfluidic qPCR(14) following off-chip cell isolation cDNA synthesis andpreamplification. In particular, microfluidic qPCR devices (BiomarkDynamic Array, Fluidigm) have recently been applied to single cellstudies (15, 16). Although these systems provide a high-throughput qPCRreadout, they do not address the front end sample preparation andrequire single cell isolation by FACS or micropipette followed byoff-chip processing and pre-amplification of starting template prior toanalysis. The critical step of integrating all steps of single cellanalysis into a robust system capable of performing measurements onlarge numbers of cells has yet to be reported. A single demonstration ofan integrated device for directly measuring gene expression in singlecells was described by Toriello et al., combining all steps of RNAcapture, PCR amplification, and end-point detection of amplicons usingintegrated capillary electrophoresis (17). Despite the engineeringcomplexity of this system, throughput was limited to four cells per run,cell capture required metabolic labeling of the cells, and the analysiswas not quantitative.

Isolation of single or limited numbers of cells is required prior tomany types of analysis and this typically requires the use of a celltrapping mechanism. Low trapping throughput and low trapping efficiencypresent a significant challenge to the goal of reliable and scalableanalysis of single or small numbers of cells. Low capture efficienciesnecessitating tens of thousands of cells in order to make a few singlecell measurements is not an issue when using cell lines, however itbecomes a significant problem when using primary samples of rare celltypes, such as stem cells. Also, observations of both the trapped cellsand those passing around the traps have indicated that the cell trappingefficiency was dependent on cell size, which could potentially introducea bias into the single cell measurements.

SUMMARY OF THE INVENTION

The present invention is based in part on the discovery that anintegrated microfluidic device may be used for high-throughput analysisof hundreds (thousands of single cells per experiment in a scalable,high performance, cost effective, and sensitive assay in pL to nLvolumes (50 pL to 100 nL) as compared to assays in μL volumes. Thedescription and examples provide the first implementation of robust andhigh-throughput single cell processing and amplification of nucleicacids on a chip, thereby achieving a major milestone in microfluidicbiological analysis.

Microfluidic technologies capable of scalable and quantitative singlecell genetic analysis are provided herein. Specifically, exemplifiedherein is an integrated microfluidic device for high-throughput RT-qPCRanalysis of mRNA and miRNA expression at a throughput of hundreds tothousands of single cells per experiment. The description shows thatthis technology provides a powerful tool for scalable single cell geneexpression measurements with improved performance, reduced cost, andhigher sensitivity as compared to analysis in μL volumes.

The examples provided herein disclose a fully integrated microfluidicdevice capable of performing high-precision RT-qPCR measurements of geneexpression from hundreds of single cells per run. Furthermore,embodiments of the device are capable of executing all steps of singlecell processing including cell capture, cell lysis, reversetranscription, and quantitative PCR. In addition to higher throughputand reduced cost, it is shown herein that nanoliter volume processingreduced measurement noise, increased sensitivity, and provided singlenucleotide specificity. The description shows an application of thistechnology to 3300 single cell measurements of i) miRNA expression inK562 cells, ii) co-regulation of a miRNA and one of its targettranscripts during differentiation in embryonic stem cells, and iii)single nucleotide variant detection in primary lobular breast cancercells. The core functionality established here provides the foundationfrom which a variety of on-chip single cell transcription analyses willbe developed. Other aspects and features of the present invention willbecome apparent to those ordinarily skilled in the art upon review ofthe following description of specific embodiments of the invention inconjunction with the accompanying figures.

Alternatively, the types of cell processing and assaying may be selectedfrom one or more of the following: rolling circle amplification,multiple displacement amplification, isothermal DNA and RNAamplifications, rapid amplification of cDNA ends (RACE), degenerateoligo primer PCR, mitochondrial DNA PCR; genomic PCR; digital PCR;RT-PCR; sequencing; immunochemistry; proximity ligation PCR, immuno PCR,metabolite analysis, enzymatic assays, reporter expression analysis;hybridization studies

According to a first embodiment there is provided, a microfluidic deviceincluding: (a) a cell capture chamber, having at least one inlet and atleast one outlet, wherein each inlet and outlet has an open and a closedposition, whereby when the inlet is in the open position fluid is ableto flow into the cell capture chamber and when the outlet is in the openposition fluid is able to flow out of the cell capture chamber, andwhereby when the inlet is in the closed position fluid is prevented fromflowing into the cell capture chamber and when the outlet is in theclosed position fluid is prevented from flowing out of the cell capturechamber, and wherein the direction of flow through the cell capturechamber dictates an upstream and a downstream orientation of the cellcapture chamber; (b) a cell funnel positioned within the cell capturechamber and operable to direct a cell passing through the cell capturechamber towards one or more desired locations within the cell capturechamber; and (c) a cell trap, positioned generally downstream from thecell funnel, whereby the cell trap is positioned to receive a cellflowing downstream from the cell funnel.

The microfluidic device may further include: (a) a second inlet in fluidcommunication with a fluid injection channel, the upstream inlet havingan open and a closed position, wherein the open position allows forfluid to enter the cell capture chamber from the fluid injection channeland in the closed position prevents fluid flow into the cell capturechamber from the fluid injection channel; and (b) a second outlet influid communication with one or more auxiliary chambers, the downstreamoutlet having an open and a closed position, wherein the open positionallows for fluid to exit the cell capture chamber and enter the one ormore auxiliary chambers and in the closed position prevents fluid flowinto the one or more auxiliary chambers from the cell capture chamber.

The microfluidic device may further include a second inlet in fluidcommunication with a fluid injection channel, the second inlet having anopen and a closed position, wherein the open position allows for fluidto enter the cell capture chamber from the fluid injection channel andin the closed position prevents fluid flow into the cell capture chamberfrom the fluid injection channel, and wherein the volume of the cellcapture chamber is expandable.

The cell capture chamber may have one inlet and one outlet, wherein theinlet is in fluid communication with a fluid injection channel, andwherein the volume of the cell capture chamber is expandable.

The cell funnel may exert a force to direct cells towards the one ormore desired locations within the cell capture chamber. The forceexerted on the cells by the cell funnel may be selected from one or moreof the following: a mechanical force; a gravitational force; anelectromagnetic force; an electrostatic force; a magnetic force; anacoustic force; a hydrodynamic force; and an optical force. Furthermore,one or more of the above forces may contribute to directing cells to adesired location. For example, a physical structure may exert amechanical force on a fluid, which in turn produces a hydrodynamicforce. The force exerted on the cells by the cell funnel may be ahydrodynamic force.

The cell trap may be selected from one or more of the following: amechanical trap; a hydrodynamic trap; a dielectrophoretic trap; amagnetic trap; an acoustic trap; an affinity trap; an optical trap; anda patch clamp trap. The cell trap may be a hydrodynamic trap.

The second outlet may be in fluid communication with a first auxiliarychamber. The first auxiliary chamber may be in fluid communication witha second auxiliary chamber, wherein there is a valve between the firstand second auxiliary chambers, wherein the valve has an open position toallow fluid flow from the first auxiliary chamber to the secondauxiliary chamber and a closed position to prevent fluid flow from thefirst auxiliary chamber to the second auxiliary chamber. The firstauxiliary chamber may be in fluid communication with a second auxiliarychamber and the second auxiliary chamber is in fluid communication witha third auxiliary chamber, wherein there is a valve between the firstand second auxiliary chambers, wherein the valve has an open position toallow fluid flow from the first auxiliary chamber to the secondauxiliary chamber and a closed position to prevent fluid flow from thefirst auxiliary chamber to the second auxiliary chamber, wherein thereis a valve between the second and third auxiliary chambers, wherein thevalve has an open position to allow fluid flow from the second auxiliarychamber to the third auxiliary chamber and a closed position to preventfluid flow from the second auxiliary chamber to the third auxiliarychamber.

The volume of the auxiliary chambers may be expandable. The volume ofthe cell capture chamber may be between 0.1 nL to 100.0 nL. Theunexpanded volume of the expandable cell capture chamber may be between0.1 nL to 100.0 nL. The volume of the cell capture chamber may be 0.6nL. The unexpanded cell capture chamber may be 0.6 nL. The effectivevolume of a given chamber may be increased by expanding the initialchamber or by opening a valve to provide fluid flow into one or moreauxiliary chambers. The ratio between the second auxiliary chamber andthe first auxiliary chamber may be 5:1. The ratio between the secondauxiliary chamber and the first auxiliary chamber may be at least 5:1.The ratio between the expanded cell capture chamber and the unexpandedcell capture chamber may be 5:1 or the ratio between the expanded firstauxiliary chamber unexpanded first auxiliary chamber may be 5:1. Theratio between the expanded cell capture chamber and the unexpanded cellcapture chamber may be at least 5:1 or the ratio between the expandedfirst auxiliary chamber unexpanded first auxiliary chamber may be atleast 5:1. The ratio between the second auxiliary chamber and the firstauxiliary chamber, or between the expanded cell capture chamber and theunexpanded cell capture chamber, or between the expanded first auxiliarychamber unexpanded first auxiliary chamber may vary depending on thereaction mixtures chosen, the concentrations of the components of themixture and the concentration of the material being assayed.Alternatively, the cell capture chamber may be between 0.05 nL and 100.0nL. Alternatively, the cell capture chamber may be between 0.05 nL and90.0 nL. Alternatively, the cell capture chamber may be between 0.1 nLand 95.0 nL. Alternatively, the cell capture chamber may be between 0.1nL and 90.0 nL. Alternatively, the cell capture chamber may be between0.1 nL and 85.0 nL. Alternatively, the cell capture chamber may bebetween 0.1 nL and 80.0 nL. Alternatively, the cell capture chamber maybe between 0.1 nL and 75.0 nL. Alternatively, the cell capture chambermay be between 0.1 nL and 70.0 nL. Alternatively, the cell capturechamber may be between 0.1 nL and 65.0 nL. Alternatively, the cellcapture chamber may be between 0.1 nL and 60.0 nL. Alternatively, thecell capture chamber may be between 0.1 nL and 55.0 nL. Alternatively,the cell capture chamber may be between 0.1 nL and 50.0 nL.Alternatively, the cell capture chamber may be between 0.1 nL and 45.0nL. Alternatively, the cell capture chamber may be between 0.1 nL and40.0 nL. Alternatively, the cell capture chamber may be between 0.1 nLand 35.0 nL. Alternatively, the cell capture chamber may be between 0.1nL and 30.0 nL. Alternatively, the cell capture chamber may be between0.1 nL and 25.0 nL. Alternatively, the cell capture chamber may bebetween 0.1 nL and 20.0 nL. Alternatively, the cell capture chamber maybe between 0.1 nL and 15.0 nL. Alternatively, the cell capture chambermay be between 0.1 nL and 10.0 nL. Alternatively, the cell capturechamber may be between 0.1 nL and 9.0 nL. Alternatively, the cellcapture chamber may be between 0.1 nL and 8.0 nL. Alternatively, thecell capture chamber may be between 0.1 nL and 7.0 nL. Alternatively,the cell capture chamber may be between 0.1 nL and 6.0 nL.Alternatively, the cell capture chamber may be between 0.1 nL and 5.0nL. Alternatively, the cell capture chamber may be between 0.1 nL and4.0 nL. Alternatively, the cell capture chamber may be between 0.1 nLand 3.0 nL. Alternatively, the cell capture chamber may be between 0.1nL and 2.0 nL. Alternatively, the cell capture chamber may be between0.1 nL and 1.0 nL.

The device may include between 1-10 cell traps and corresponding cellfunnels. Alternatively, a funnel may be used in conjunction with morethan one dedicated trap. The cell traps may also be designed to holdmore than one cell each and each may be fed by one or more cell funnels.Additionally, cell traps may be sized such that they select forparticular cell types if a mixed population of cells is being assayed.Furthermore, the cell traps and funnels may be designed to excludecertain cell types or to select certain cell types.

The cell funnel may include a pair of cell deflectors each having aproximal and a distal end, wherein the proximal ends are positioned atopposite sides of the capture chamber, and wherein each distal end ofthe cell deflector is angled on the diagonal in a downstream directionrelative to the proximal ends, whereby the distal ends of the celldeflectors provide an opening sized to permit the passage of a cellbetween the distal ends of the cell deflectors. The cell trap may begenerally cup or “U” shaped, or have a region that is generally cup or“U” shaped such that cells may enter the interior of the “U” shape fromone side but not pass through. The cell trap may provide for fluid flowthrough the cell trap. The flow through of the trap may be before,during and after a cell is captured in the trap. Such flow through mayassist with capturing the cell and with washing of a captured cell.Furthermore, the cell funnel or the cell trap may have a structure asshown in any one of FIGS. 1 a-x, 2 a-e, 12 b-i, and 24.

According to a further embodiment there is provided, a cell capture andpreparation method, the method including: (a) flowing cells in a fluidthrough a chamber; (b) funneling the cells in the fluid towards a celltrap; (c) capturing a predefined number of the cells within the chamber;(d) interrupting the flow of cells in the fluid; (e) washing thecaptured cells by flowing a wash solution through the chamber, whereinthe flow of wash solution removes contaminants from the chamber; and (f)sealing the predefined number of cells in the chamber.

According to a further embodiment there is provided, a cell capture andpreparation method, the method including: (a) flowing cells in a fluidthrough a chamber; (b) funneling the cells in the fluid towards a celltrap; (c) capturing a predefined number of the cells within the chamber;(d) interrupting the flow of cells in the fluid; (e) washing thecaptured cells by flowing a wash solution through the chamber, whereinthe flow of wash solution removes contaminants from the chamber; and (f)sealing the predefined number of cells in the chamber.

The method may further include processing and assaying the washedcaptured cells, wherein assaying is selected from one or more of thefollowing: rolling circle amplification, multiple displacementamplification, isothermal DNA and RNA amplifications, rapidamplification of cDNA ends (RACE), degenerate oligo primer PCR,mitochondrial DNA PCR; genomic PCR; digital PCR; RT-PCR; sequencing;immunochemistry; proximity ligation PCR, immuno PCR, metaboliteanalysis, enzymatic assays, reporter expression analysis; hybridizationstudies, etc.

The method may further include: (a) cell lysis; (b) reversetranscription; and (c) amplification.

The method may further include: (a) cell lysis; (b) reversetranscription; and (c) quantitative amplification.

According to a further embodiment there is provided, a cell assaymethod, the method including: (a) directing cells to a cell capturechamber, wherein the chamber has a volume of between 0.1 nL to 100 nL;(b) capturing a predefined number of the cells within the chamber; (c)washing the captured cells by flowing a wash solution through thechamber, wherein the flow of wash solution removes contaminants from thechamber; and (d) isolating the predefined number of cells in thechamber.

The method may further include assaying the washed captured cells,wherein assaying is selected from one or more of the following: rollingcircle amplification, multiple displacement amplification, isothermalDNA and RNA amplifications, rapid amplification of cDNA ends (RACE),degenerate oligo primer PCR, mitochondrial DNA PCR; genomic PCR; digitalPCR; RT-PCR; sequencing; immunochemistry; proximity ligation PCR, immunoPCR, metabolite analysis, enzymatic assays, reporter expressionanalysis; hybridization studies, etc.

The method may further include: (a) cell lysis; (b) reversetranscription; and (c) amplification.

The method may further include: (a) cell lysis; (b) reversetranscription; and (c) quantitative amplification.

According to a further embodiment there is provided, a cell assaymethod, the method including: (a) directing cells to a cell capturechamber, wherein the chamber has a volume of between 0.1 nL to 100 nL;(b) capturing a predefined number of the cells within the chamber; (c)washing the captured cells by flowing a wash solution through thechamber, wherein the flow of wash solution removes contaminants from thechamber; (d) isolating the predefined number of cells in the chamber;(e) lysing the cells within the chamber; (f) reverse transcribing theRNA released by the cell lysis; and (g) amplifying the cDNAs transcribedin (f) by polymerase chain reaction (PCR).

According to a further embodiment there is provided, a cell assaymethod, the method including: (a) directing cells to a cell capturechamber, wherein the chamber has a volume of between 0.1 nL to 100 nL;(b) capturing a predefined number of the cells within the chamber; (c)washing the captured cells by flowing a wash solution through thechamber, wherein the flow of wash solution removes contaminants from thechamber; (d) isolating the predefined number of cells in the chamber;(e) lysing the cells within the chamber; (f) reverse transcribing theRNA released by the cell lysis; and (g) amplifying the cDNAs transcribedin (f) by quantitative polymerase chain reaction (PCR).

According to a further embodiment there is provided, a cell capture andpreparation method, the method including: (a) flowing cells in a fluidthrough a chamber; (b) funneling the cells in the fluid towards a celltrap; (c) capturing a predefined number of the cells within the chamber;(d) interrupting the flow of cells in the fluid; and (e) sealing thepredefined number of cells in the chamber.

According to a further embodiment there is provided, a cell capture andpreparation method, the method including: (a) flowing cells in a fluidthrough a chamber; (b) funneling the cells in the fluid towards a celltrap; (c) capturing a predefined number of the cells within the chamber;(d) interrupting the flow of cells in the fluid; and (e) sealing thepredefined number of cells in the chamber.

According to a further embodiment there is provided, a cell assaymethod, the method including: (a) directing cells to a cell capturechamber, wherein the chamber has a volume of between 0.1 nL to 100 nL;(b) capturing a predefined number of the cells within the chamber; and(c) isolating the predefined number of cells in the chamber.

According to a further embodiment there is provided, a cell assaymethod, the method including: (a) directing cells to a cell capturechamber, wherein the chamber has a volume of between 0.1 nL to 100 nL;(b) capturing a predefined number of the cells within the chamber; (c)isolating the predefined number of cells in the chamber; (d) lysing thecells within the chamber; (e) reverse transcribing the RNA released bythe cell lysis; and (f) amplifying the cDNAs transcribed in (e) bypolymerase chain reaction (PCR).

According to a further embodiment there is provided, a cell assaymethod, the method including: (a) directing cells to a cell capturechamber, wherein the chamber has a volume of between 0.1 nL to 100 nL;(b) capturing a predefined number of the cells within the chamber; (c)isolating the predefined number of cells in the chamber; (d) lysing thecells within the chamber; (e) reverse transcribing the RNA released bythe cell lysis; and (f) amplifying the cDNAs transcribed in (e) byquantitative polymerase chain reaction (PCR).

The method may further include a cell sorting step prior to directingthe cells to a cell capture chamber. The method may further includeimmobilizing the captured cell prior to washing. Immobilization mayfurther enhance washing of the cells in the capture chamber.

BRIEF DESCRIPTION OF THE DRAWINGS

In drawings which illustrate embodiments of the invention,

FIG. 1 a-l are schematic drawings of a cell funnel and correspondingcell cages according to various embodiments of the invention involvinghydrodynamic cell traps.

FIG. 1 b are schematic drawings of cell cages according to variousembodiments of the invention involving hydrodynamic cell traps.

FIG. 2 a is a schematic drawing a of cell capture testing device.

FIG. 2 b is a schematic drawing of a cell funnel and cell trap geometryaccording to one embodiment of the invention.

FIG. 2 c is a schematic of a preferred cell funnel and cell trapgeometry according to one embodiment of the invention.

FIG. 2 d is optical micrograph of a single K562 approaching a trap withthe geometry presented in FIG. 2 c.

FIG. 2 d is optical micrograph of the K562 in FIG. 2 c after beingtrapped. 2 c.

FIG. 3 is a graph depicting the results of a comparison of chemical andheat lysis methods.

FIG. 4 is a graph comparing background signal to signal from a singlecell.

FIG. 5 is a graph depicting the effect of cell lysate concentration oninhibition of RT-qPCR.

FIG. 6 is a graph showing the effect of different RT to PCR dilutions onqPCR performance.

FIG. 7 is schematic drawing for a microfluidic device according to oneembodiment of the invention.

FIG. 8 is a graph depicting qPCR results on nine single cells assayedfor miRNA16 and one no cell control.

FIG. 9 is a graph depicting elution efficiency measurement of three ofthe chambers from the experiment presented in section after being elutedtwice.

FIG. 10 is a schematic diagram of a microfluidic device for single cellcapture analysis according to one embodiment of the invention.

FIG. 11 is a schematic diagram outlining the operation of themicrofluidic device of FIG. 10.

FIG. 12A is a microfluidic device according to one embodiment of theinvention. Scale bar: 4 mm. The device features 6 sample input channels,each divided into 50 compound reaction chambers for a total of 300RT-qPCR reactions using approximately 20 μL of reagents. Rectangular boxindicates the region depicted in B.

FIG. 12B is an optical micrograph of the array unit corresponding to thearea within the black rectangle in FIG. 12A.

FIG. 12C is an optical micrograph of two cell capture chambers withtrapped single cells.

FIG. 12D is schematic drawing of a single cell suspension injected intothe device depicted in FIG. 12A

FIG. 12E is schematic drawing of a cell trap isolating a single cellfrom the fluid stream to permit washing of the cell.

FIG. 12F is schematic drawing of actuation of pneumatic valves to resultin single cell isolation prior to heat lysis.

FIG. 12G is schematic drawing of injection of reagent for reversetranscription (RT) reaction.

FIG. 12H is schematic drawing of the reagent injection line beingflushed with subsequent reagent for PCR.

FIG. 12I is schematic drawing of reagent for qPCR being combined with RTproduct in a 50 nL qPCR chamber.

FIG. 13A is a fluorescence image of entire device of FIG. 12A showing300 reactions in 6 lanes.

FIG. 13B is a graph of 300 real time amplification curves generated fromprocessing sequences of images similar to that in FIG. 12A.

FIG. 13C is a graph of on-chip and off-chip RT-qPCR for GAPDH from a 8×serial dilution of purified total RNA.

FIG. 13D is a graph depicting the real-time amplification curves ofGAPDH in K562 cell lysate dilutions.

FIG. 13E is a graph depicting the measured CT values for GAPDH indilution series of cell lysate.

FIG. 14 is a graph comparing GAPDH measurements from K562 cell lysatewith RT performed in the microfluidic device of FIG. 12A, or RTperformed in tubes prior to qPCR in the device.

FIG. 15A is schematic drawing of the locations of cells in each chamberalong all 6 lanes of a device of FIG. 12A.

FIG. 15B is a scatter plot showing CT measurements for experiment shownin FIG. 14A.

FIG. 15C is a histogram of the number of GAPDH transcripts measured insingle K562 cells (N=233).

FIG. 16A is a histogram showing the size distribution of cells inoriginal sample as measured by Cedex.

FIG. 16B is a histogram showing the size distribution of cells isolatedby microfluidic traps.

FIG. 17A is a scatterplot of GAPDH measurements in cells washed in PBSoff-chip prior to injection into microfluidic device.

FIG. 17B is a scatterplot of GAPDH measurements in cells washed in PBSon-chip prior to injection into microfluidic device.

FIG. 17C is a graph of GAPDH measurements from loading purified RNA andwashing, or not washing, the cell capture chambers.

FIG. 18A is a schematic diagram of the locations of cells in eachchamber along all 6 lanes of a device.

FIG. 18B is a fluorescence image of entire device, corresponding toexperiment in FIG. 18A) after 30 PCR cycles.

FIG. 19 is a scatterplot of miR16 measurements in hESC cell aggregates.

FIG. 20A is a histogram of single cell measurements of miR-16 expressionin K562 cells and hESCs.

FIG. 20B is a histogram of differential expression of miR-223 betweenK562 cells and hESCs.

FIG. 20C is a graph of mean single cell miRNA copy numbers measured byRT-qPCR in a microfluidic device according to one embodiment of theinvention compared to digital PCR measurements from bulk cell lysate

FIG. 20D is a histogram of differential expression of miR-223 betweenK562 cells and hESCs.

FIG. 21A is a histogram of single cell measurements of miR-16 expressionin K562 cells and hESCs.

FIG. 21B,C are histograms showing the distribution of miR-145 and OCT4transcripts.

FIG. 21D is a scatterplot showing co-expression measurements of SP1wild-type and SNV mutant transcripts in primary cells isolated from alobular breast cancer sample

FIG. 22A is a representative image of mRNA-FISH of OCT4 in a CA1S cellafter 7 days of FBS differentiation

FIG. 22B is a representative image of undifferentiated CA1S cells.

FIG. 23 is a histogram showing the percentage of aligned tags at eachtag length in a miRNA sequence tag library constructed according to oneembodiment of the invention.

FIG. 24 is a schematic diagram of a microfludic device for sequencingand characterizing a single cell transcriptome according to oneembodiment of the invention.

FIG. 25A is an image illustrating the mixing of functionalized beads anda cell for the purpose of purifying and immobilizing template (RNA)according to an embodiment of the invention.

FIG. 25B is an image illustrating the stacking of functionalized beadsin a chamber according to one embodiment of the invention.

FIG. 26 is an of a microfluidic device for magnetic immobilization offunctionalized beads to bind template (DNA/RNA) and enable multi-stepreactions in a single chamber according to an embodiment of theinvention.

FIG. 27 is a schematic diagram of a microfluidic device integratingsingle cell processing with microarray spotting to address differentreagents to different reactions.

FIG. 28 is a schematic diagram of a microfluidic device for integratedsingle cell digital PCR according to one embodiment of the invention.

FIG. 29 is a schematic diagram of the microfluidic device of FIG. 28,depicting the mixing of the PCR solution and template molecules by abifurcating rotary mixer.

FIG. 30 is a schematic diagram of a microfluidic device for sequentiallydiluting cell products by passing them to larger chambers according toone embodiment of the invention

FIG. 31 is a schematic diagram of a microfluidic device for multiplexedsingle cell RT-qPCR according to one embodiment of the invention.

DETAILED DESCRIPTION

A variety of techniques have been developed in order to isolate andmeasure single cells. These techniques all greatly differ in both thenumber of cells they are able to analyze and the number of parallel, ormultiplexed, measurements they are able to perform.

DEFINITIONS

“Flow cytometry”, as used herein, is a widespread technique for rapidlyand quantitatively examining and sorting populations of cells,organelles and other constituents based on single particle measurementsof fluorescence, light scattering and optical absorbance (18). Flowcytometry is able to process and measure up to hundreds of thousands ofcells per minute, using optical multiplexing to measure differentattributes. This technique has been used to measure both physicalproperties and chemical composition of single cells (19). Measuring bothmRNA (20) and miRNA (21) expression using flow cytometry has beenperformed by coupling transcriptional reporters such as GFP to genes ormiRNA of interest and recording the fluorescence of each cell. Sortingsingle cells via flow cytometry for further genetic analysis via RT-qPCRhas also been previously reported (22).

“Laser capture microdissection” (LCM), a used herein, is a technique inwhich pure populations from heterogeneous samples under directvisualization can be isolated for further analysis (23). This techniquecan be used on tissues (23), cytological preparations (24) and live cellculture (25). In LCM, cells of interest are visualized under amicroscope and the user selects which regions to isolate from the restof the sample. These selected regions can then be isolated from the restof the sample by either ablating the surrounding regions, or cutting outthe desired sections from the sample. Because this method isnon-destructive to the samples being isolated, cells isolated via thismethod can be measured using any traditional molecular biologytechniques.

A “microfluidic device”, as used herein, refers to any device thatallows for the precise control and manipulation of fluids that aregeometrically constrained to structures in which at least one dimension(width, length, height) is less than 1 mm.

A “cell capture chamber”, as used herein, refers to an enclosed spacewithin a microfluidic device in which one or more cells may be isolatedfrom a larger population of cells as the cells are flowed through thedevice. Each cell capture chamber will have at least one inlet forpermitting fluid, including fluid containing cells, to enter thechamber, and at least one outlet to permit fluid to exit the chamber.Persons skilled in the art will understand that an inlet or an outletcan vary considerably in terms of structure and dimension, and may becharacterized in a most general sense as an aperture that can bereversibly switched between an open position, to permit fluid to flowinto or out of the cell capture chamber, and a closed position to sealthe cell capture chamber and thereby isolate and retain its contents,whereby the aperture may also be intermediate between the open andclosed positions to allow some fluid flow.

The direction of fluid flow through the cell capture chamber dictates an“upstream” and a “downstream” orientation of the cell capture chamber.Accordingly, an inlet will be located at an upstream position of thechamber, and an outlet will be generally located at a downstreamposition of the chamber. A person skilled in the art will understand,however, that a single aperture could function as both an inlet and anoutlet.

An “inlet” or an “outlet”, as used herein, may include any or anaperture whereby fluid flow is restricted through the inlet, outlet oraperture. There may be a valve to control flow, or flow may becontrolled by separating the channels with a layer which prevents flow(for example, oil).

A “cell capture chamber”, may further include a “cell trap” positionedgenerally downstream from the “cell funnel”, whereby the cell trap ispositioned to receive (and retain) a cell flowing downstream from thecell funnel.

A “cell funnel”, as used herein, refers to an apparatus which isdesigned to focus the flow of cells from an upstream location, where thecells are dispersed, to one or more desired downstream locations withinthe cell capture chamber having a smaller cross sectional area of cellflow. The cell funnel exerts a force to direct cells towards the one ormore desired locations within the cell capture chamber. For the purposesof clarity, “force” is defined herein as any influence that causes afree body (e.g. a cell) to undergo a change in velocity. Funnels mayeither span the entire height and/or width of the cell capture chamber,or partially span the height and/or width.

FIG. 1 a-l provides illustrations of exemplary mechanical funnelsaccording to various embodiments of the invention. The basic mechanicalfunnel 10 of FIG. 1 a can be modified by making a constriction 12 longeras in FIGS. 1 b and e. There may be multiple funnels, such as the nestedfunnels 10 a and 10 b in FIG. 1 d, i, and j, and 10 c in FIG. 1 f. Theangle θ of the funnels may be varied as exemplified in FIG. 1 k. Theconstriction 12 of the funnel may be straight as in FIGS. 1 d, e, f, g,k, or angled as in FIGS. 1 a, b, h, i, j, k, l. Funneling may beadditionally accomplished through a combination of mechanical andhydrodynamic means as in FIG. 1 c (i.e. a different fluid, or the samefluid at a different flow rate, may flow in through the outsidechannels). A funnel may also comprise a change in the cross-section ofthe upstream channel, such as a groove, that causes cells to bepositioned at a specified section along the width of the channel priorto encountering the cell trap.

A “cell trap”, as used herein, refers generally to a means for receivingand retaining cells at a pre-determined location over time. A cell trapmay comprise localized surface modifications for chemical immobilizationof a cell. Alternatively, the cell trap may be a mechanical trap, ahydrodynamic trap (10, 26-28), a hydrodynamic balancing trap (29, 30),an active valving trap (2, 10, 31, 32), a dielectrophoretic trap (33), aDNA immobilization trap (17), a gel encapsulation trap (34), a magnetictrap, an acoustic trap or an optical trap (35). In various embodimentsof the current invention, a cell trap will generally be positioneddirectly in the path of the smaller cross sectional of cell flow createdby the funnel. Where a mechanical funnel as illustrated in FIG. 1 isused according to various embodiments of the invention, a trap may bepositioned directly after the downstream opening of the funnel.

A “mechanical trap”, as used herein, refers to a physical cell trap suchas a cage.

A “hydrodynamic trap”, as used herein, refers to a cell trap in whichthe force of the fluid in motion plays a role in retaining a trappedcell in its position. A hydrodynamic trap may be also be comprised of amechanical trap in which a cell is captured and retained. In certainembodiments of the invention utilizing hydrodynamic traps, it may bedesirable to have three or more inlets to the cell capture chamber sothat the flows may be adjusted in order to direct cells to the traps.

A “dielectrophoretic trap”, as used herein, refers to a cell trap inwhich cells, being dielectric objects, are retained by the forcesgenerated by a non-uniform electric field.

A “magnetic trap”, as used herein, refers to a cell trap employingmagnetic fields to retain cells. Typically, cells will be labeled withmagnetic particles, and then positioned and retained by the magneticfields. However, magnetic traps can also be used to trap-non-magneticcells in suitable buffers.

An “acoustic trap”, as used herein, refers to a cell trap in whichultrasonic standing waves are used to generate stationary pressuregradients that exert forces that position and retain cells.

An “optical trap”, as used herein, refers to a cell trap in which atightly focused laser beam, typically a near-infra red laser beam, isused to draw cells in the direction of the beam.

The size of the cell trap may be varied according to the size, type,mechanical properties, or number of cells that is desired to be trapped.A microfluidic device according to various embodiments of the inventionmay further include a combination of trap designs for the capture of arange of cell types. Furthermore, each cell capture chamber couldinclude multiple traps. In such embodiments, the frequency of cells ofthat are trapped at each size could be used as a diagnostic.Alternatively, the contents of a group of cells caught in a single trapcould be processed and analyzed.

FIG. 1 m-x provides illustrations of exemplary physical cages used inhydrodynamic traps according to various embodiments of the invention.The inner shape of the cages may be cup-shaped, as are the cages 20 inFIG. 1 m-x, to accommodate spherical-shaped cells, or may be designedwith various geometries to accommodate cells of unique shape and/orsize. The cages may form weirs that span essentially the entire heightor width of the chamber, or partially span the height or width of thechamber, or both. The outer shape of the cage may be changed to modulatefluid flow around the trap as in FIG. 1 m. Perforations or sieveelements 22 can be added as in FIGS. 1 o, p, q, r, s, and x tofacilitate and modulate fluid flow through the cup. The perforation sizemay be decreased or increased as in FIG. 1 x. The perforations mayeither be the entire height or width of the chamber, or partially spanthe height or width of the chamber, or both. The size of the cage can bemade longer as in FIG. 1 t, wider as in FIG. 1 u, longer and wider as inFIG. 1 v, or shorter and narrower as in FIG. 1 w.

The physical structures forming the funnel may be angled on both sides,i.e. also on the side opposite the funneling surface, to facilitateremoving cells from the trap, as with angles ψ in FIGS. 1 k and l.Further features, in addition to the trap, may be located downstream ofa funnel to further modulate the fluidic impedance, such as elements 14in FIGS. 1 h and 1 l.

A “fluid injection channel”, as used herein, refers to any conduitthrough which fluid may be introduced into a chamber of the device. Afluid injection channel can be used to deliver any fluid to a chamberincluding cell suspensions, wash buffers, reaction mixes, etc.

An “auxiliary chamber”, as used herein, refers to any chamber subsidiaryto a cell capture chamber. Auxiliary chamber can be used for treatmentor assaying of a captured cell, or its isolated contents. Treatment caninclude cell preparation steps including culture, washing, lysis, andfractionation. Assaying may include DNA and RNA amplification anddetection, including mitochondrial PCR; genomic PCR; digital PCR,RT-PCR, RTq-PCR, multiple displacement amplification (DNA), rollingcircle amplification sequencing, degenerate PCR, molecular inversionprobes, molecular beacons, as well as other DNA/RNA amplification anddetection methods, in vitro transcription, ligation, immunochemistry;reporter expression analysis; hybridization studies; and so forth.Several auxiliary chambers may be connected, in tandem and/or inparallel, to a single cell capture chamber, such that multipletreatments may be performed on the contents of a single cell capturechamber. A valve may be positioned between an auxiliary chamber and thecell capture chamber, or between auxiliary chambers, to regulated fluidflow between chambers.

An “expandable” cell capture chamber, as used herein, refers to a cellcapture chamber that may be expanded, or contracted, during operation toaccommodate different fluid volumes. As such, a single chamber can beused for the purposes of cell capture and subsequent treatment andanalysis that must be conducted in different fluid volumes, and therebyavoid the need for multiple discrete chambers.

Expansion of an expandable cell capture chamber may be effected inseveral ways. For example, the chamber may comprise a syringe in whichchamber volume is modulated by use of a plunger. Alternatively, thechamber may be constructed of resilient materials that can expand withthe addition of fluid, or contract when the fluid volume is released.Yet alternatively, the chamber may be partially defined by an immiscibleliquid (such as air or oil), such that a greater fluid volume may beaccommodated by movement of the liquid boundary.

A person skilled in the art will understand that an auxiliary chambermay be expandable. Accordingly, a microfluidic device may be comprisedof a combination of non-expandable and/or expandable cell capturechambers and auxiliary chambers.

“Contaminants”, as used herein, refers to any material that mayinterfere with the precision and/or accuracy of the assays of the cellor cell contents. Contaminants include, but are not limited to proteins,small molecules, salts, buffers, RNA, DNA, other cells, particles, andso forth.

“MicroRNAs” (miRNA), as used herein, are short (19-23 nucleotides long)non-coding ribonucleic acid (RNA) polymers that are implicated in thepost-transcriptional regulation of the translation of complementary mRNAinto proteins (36). They are expressed as larger transcripts that formself-complementary “hairpin” RNA. miRNA have been shown to be central tomany biological pathways including cell proliferation (37),differentiation (38) and death (39), developmental timing (40) andpatterning (41), nervous system patterning (39, 42), and virusresistance (43, 44). The wide variety of functional effects, combinedwith the knowledge that the miRNA expression profile is a uniquesignature of cellular state (45) suggests that insight into thecomplicated biochemical nature of many diseases, such as cancer (45-47)and heart disease (48-51), can be gained through studying miRNAexpression.

“Reverse transcription quantitative polymerase chain reaction” (RT-qPCR)is a molecular biology technique that is commonly used to measure geneexpression (52-54). RT-qPCR extends the functionality of the traditionalpolymerase chain reaction (PCR), which is a method of specifically andexponentially amplifying a single or a few copies of DNA in situ (54).In RT-qPCR, the first strand of DNA is synthesized from a RNA templatethrough a process called reverse transcription (RT; 54). Specificity isadded to the reverse transcription step by exploiting the RT enzymes'need to have a DNA-RNA duplex. Oligonucleotides called primers designedto be complementary to the desired target are thus used to specificallytranscribe a piece of RNA into DNA. After the first strand synthesis,traditional PCR is performed in the presence of a fluorescent reporterand primers designed to amplify the DNA transcribed in the previous step(54). The reaction is then monitored after every cycle, and thefluorescence is recorded, producing characteristic sigmoidal curves.After the completion of the reaction, it is then possible to determinethe relative starting abundances of the genes (54). The use of RT-qPCRto measure single cell mRNA (55, 56) and miRNA (57, 58) expressionlevels is well established. It will be understood by those skilled inthe art that the analysis of more than one sequence in a single reactionmay be performed using optical multiplexing strategies. It will furtherbe understood by one skilled in the art that in addition toquantification of defined sequences, RT-PCR may be used to amplify oneor more genes for subsequent recovery and/or analysis.

While the embodiments of the invention described herein are generallyconcerned with the capture and isolation of cells, and subsequentprocessing, it will be appreciated that the microfluidic devicesaccording to various aspects of the invention could be used for thecapture and analysis pre-determined numbers of entities other thancells, including cell organelles, viruses, microparticles, droplets,etc.

Example 1 Example 1.1 Materials and Methods Nucleic Acid Detection andQuantification

DNA quantification through microarray analysis is a highly multiplexed,well established method to assay a sample for thousands of differenttargets. Specific DNA sequences that have been immobilized on a solidsurface act as probes to target molecules in solution. The solutioncontaining these target molecules is flowed over the surface of themicroarray, and target molecules bind to the immobilized probes viastandard DNA base pairing. Probe-target hybridization is detected andquantified by the detection of a fluorophore or a silver orchemiluminescent target. While microarray analysis has been used onsingle cells, it requires an amplification step (59) in order togenerate the quantity of target molecules required to meet the assaydetection limits.

Single molecule imaging techniques, including fluorescence in situhybridization (FISH; 60, 61) and single fluorophore imaging (62) havebeen used to directly count the number of transcripts in a single cell.While these methods stand alone in their ability to quantify transcriptabundance by direct observation, they can only be multiplexed optically,and require highly specialized equipment.

Methods that have been developed to specifically detect and quantifymiRNA include northern blot (64), in situ hybridization (60, 61), singlemolecule imaging (62), microarray (65, 66), next-generation sequencing(67) and RT-qPCR with stem-loop RT primers and TaqMan probes (68).Because of the large dynamic range, high degree of specificity and thefact that many methods of single cell transcript quantification requirea PCR amplification, RT-qPCR using stem-loop primers and TaqMan probeswas chosen in order to measure miRNA abundance. Stem-loop primerscontain a self-complementary region, and therefore “fold back” onthemselves creating a hairpin structure. This structure prevents theprimer from binding to RNA molecules other than at the very end of amolecule, which prevents miRNA precursors from being amplified (68).TaqMan probes are hydrolysis probes designed to increase the specificityof qPCR (69). A hydrolysis probe is an oligonucleotide labeled with afluorophore on one end, and a quencher on the other. When the completeprobe is free in solution, the fluorophore is close enough to thequencher such that any fluorescence emitted by the fluorophore isquenched by the quencher through fluorescence resonance energy transfer(FRET) (69). During PCR, the probe binds to its complementary sequence,and is cleaved through the exonuclease activity of the Taq polymerase,thereby separating the fluorophore and the quencher (69). Thus, aftereach cycle, there will ideally be a two-fold increase in fluorescence.Through measuring the fluorescence after every cycle, or in “real time”,it is possible to determine relative starting abundances. The inclusionof a serial dilution of known concentrations in the experiment (referredto as a standard) produces a calibration curve, allowing the initialstarting number of molecules to be calculated.

Fabrication

Multilayer Soft Lithography (MSL) was used to fabricate devices (63,70). The fabrication process takes advantage of well-establishedphotolithography techniques and advances in microelectronic fabricationtechnology. The first step in MSL is to draw a design using computerdrafting software, which is then printed on high-resolution masks.Silicon wafers covered in photoresist are exposed to ultraviolet light,which is filtered out in certain regions by the mask. Depending onwhether the photoresist is negative or positive, either areas exposed(negative) or not (positive) will crosslink and the resist willpolymerise. The unpolymerised resist is soluble in a developer solutionand is subsequently washed away. By combining different photoresists andspin coating at different speeds, wafers can be patterned with a varietyof different shapes and heights. The wafers are then used as moulds totransfer the patterns to polydimethylsiloxane (PDMS). In MSL, stackingdifferent layers of PDMS cast from different moulds on top of each otheris used to create channels in overlapping “flow” and “control” layers(63, 70). The two (or more) layers are bound together by mixing apotting prepolymer component and a hardener component at complementarystoichiometric ratios to achieve vulcanization. In order to create asimple microfluidic chip, a “thick” layer is cast from the mouldcontaining the flow layer, and the “thin” layer is cast from the mouldcontaining the control layer. After partial vulcanization of bothlayers, the flow layer is peeled off the mould, and manually aligned tothe control layer. These layers are allowed to bond, and then thisdouble slab is peeled from the control mould, and then holes for inletsand outlets are punched and the double slab is bonded to a blank layerof PDMS. After allowing more time to bond, the completed device ismounted on glass slides.

Fluid flow in the device is controlled using off-chip computerprogrammable solenoids which actuate the pressure applied to fluid inthe control layer. When pressure is applied to these lines, the flexiblemembrane between the overlapping orthogonal control and flow linesdeflects into the flow channel, effectively valving the flow. Differentcombinations of these valves can be used to create peristaltic pumps,multiplexer controls and isolate different regions of the chip.

Example 1.2 Cell Capture

In order to increase the capture efficiency, decrease the sizeselectivity of the trap design, and attempt to characterize how trapdimensions affect capture efficiencies for different cell types, avariety of different trap geometries were designed and tested on twodifferent cell lines: K562 cells, a human erythroleukemic cell line withan average diameter of 18 microns, and nBAF3, a murine pro-B-cell linewith an average diameter of 12 microns.

Ninety-six (96) different trap geometries were fabricated and tested insix independent devices. A schematic of cell capture testing device isshown in FIG. 2 a. A single device is able to analyze 16 different trapgeometries, each replicated 83 times in a single column, with 150microns separating each trap. A binary demultiplexor (top of device) isused to select the desired trap geometry, and a microfluidic peristalticpump (bottom of device) is used to pump the cells through the device.The scale bar is 500 microns. The inset shows an enlarged regionencompassing two cell traps. The parameters tested in the screen ofgeometries were “a” the distance between the flow focuser and thecapture weir, “b” the width of the sections removed from the weir (i.e.the sieve elements), “c” the number of sections removed (i.e. sieveelements) and d the dimensions of the capture weir. The inset scale baris 100 microns. FIGS. 2 b and 2 c show the original and optimized trapgeometries, respectively. FIG. 2 d shows an optical micrograph of asingle K562 approaching the trap geometry presented in FIG. 2 c. FIG. 2e shows this same cell after being trapped. The scale bar in B-E is 100microns.

The fabrication protocol was optimized so that high aspect ratio(ranging from 7:2 to 14:1) could be reproducibly fabricated across awafer. In all cases, the channel containing the trap, in theseembodiments a capture cage forming a weir, was 14 microns high, and thefunnels consisted of two angled, 10 microns wide obstacles.

The distance between the cell trap and the funnel was varied between22.5 and 8.5 microns, corresponding to an approximate range of 1.25 to0.5× the diameter of an average K562 cell, and 1.9 to 0.7× the diameterof an average nBAF3 cell. Observations of the cells entering the cellcapturing chamber indicated the geometries with the capture cell 8.5microns from the funnel maximized the single cell fill factor. However,while this distance maximized the fill factor, an increase in the numberof blockages was also observed, and so a distance of 10.5 microns waschosen to be integrated in the final device.

The capture cage size was varied between 12×12 microns to 20×36 microns.As expected, an increase in the number of traps containing more than asingle cell was observed with increasing cup size.

The removal of one, two, three and four sections to create sieveelements with widths varying from 1 micron to 6 microns (1, 2, 4, 6) wasalso tested. Observations of cells entering the traps indicated that 6micron wide sieve elements permitted some cells to squeeze through thetrap. There were not any observable differences between removing one ortwo sections from (i.e. between creating one or two sieve elements in)the trap. In order to determine if there was enough flow through thetraps with sieve elements such that the funnels could be removed fromthe design, a selection of the traps with different numbers of sieveelements was tested without the funnels. In all these cases, no cellswere observed to be trapped in a trap that wasn't directly downstream ofa funnel.

Thus, after testing the 96 different trap geometries, satisfactorycapture efficiencies on K562 cells were observed for a 12×12 microncapture cup located 10.5 microns from the flow focusers, with two 4micron segments removed from the capture weir.

Example 1.3 Lysis

Two lysis methods were selected for their ease of integration into amicrofluidic device: heat lysis, which involves heating the sample to85° C. for 7 minutes, or a heat-inactivated chemical lysis bufferprovided in the Invitrogen SuperScript® III CellsDirect cDNA SynthesisKit. In order to assess the relative efficiency of each method onreleasing miRNA from the cells and determine if further reactions wouldbe inhibited by the chemical lysis buffer, a sample of K562 cells wasserially diluted and lysed using each method. Released miRNA from thesesamples was subsequently reverse-transcribed into cDNA and the amount ineach sample was quantified using qPCR.

The results from this test can be found in FIG. 3. The efficiency of thechemical lysis more closely resembles the expected value ofapproximately −3.3 cycles per 10-fold dilution. However, the increasedbenefit of the chemical lysis is reduced by the fact that using itrequires the addition of an extra chamber on the final device, therebysacrificing valuable density.

Example 1.4 Cell Loading and on-Chip Cell Wash

Preliminary results shown in FIG. 4 indicated that it was not possibleto distinguish between background signal and the signal from a singlecell. However, the possibility that this signal was coming solely fromreagent contamination was ruled out as a region of the chip in whichcells did not flow through was distinguishable from the rest of thebackground. This result suggested that there was a non-negligible amountof free floating miRNA in the media the cells were suspended in. Thesolution to this problem was to wash the cells captured in the trapswith clean culture media before lysis, thereby washing any free-floatingmiRNA, excess cells and any other debris out from the chip.

Example 1.5 Cell Lysate Inhibition of RT-qPCR

High concentrations of cell lysate inhibit molecular biologicalreactions. The following experiment was performed in order to determinethe optimal cell lysate dilution so that subsequent RT and qPCRreactions can be performed on the sample. A 10× dilution series of K562cell lysate prepared off-chip (10 cells to 1/1000 cell equivalents percapture chamber) was loaded into a custom microfluidic device whichcontained an array of chambers testing a variety of cell lysate to RTvolume dilution ratios. All reactions were performed in technicaltriplicate. A synthetically synthesized species of miRNA fromCaenorhabditis elegans (cel-mir-2) was added to the reversetranscription mix at a constant concentration of 0.2 ng/nL. The standardmiRNA pulsed reverse transcription 38,39 was performed, the product fromthis reaction was diluted five-fold, and on-chip qPCR was performed,assaying for the synthetic C. elegans miRNA.

Because the assayed miRNA is not found in K562 cells, the effect ofinhibition due to different concentrations of cell lysate on RT-qPCR canbe inferred. The results from this experiment are presented in FIG. 5.An increase in cycle threshold (C_(t)) was seen at concentrations of 10cells per capture chamber at 1 nL, 2 nL, 4 nL and 8 nL RT chambervolumes. This increase in C_(t) value directly corresponds to reactioninhibition due to cell lysate. Thus, in order to account for a varietyof cell sizes and types, a minimum RT chamber volume of dilution ofapproximately 13× (0.6 nL cell capture chamber into 8 nL RT chamber) ispreferred to sufficiently dilute out the cell lysate such thatdownstream RT-qPCR reactions are not inhibited.

Example 1.6 RT to PCR Dilution

RT to PCR dilution ratios of 1:2.1 to 1:21 were tested in order todetermine the effect of this dilution on qPCR performance. K562 lysatewas processed in tubes, reverse transcribed, and added in differentdilutions to PCR mix.

FIG. 6 shows the effect of different RT to PCR dilutions on qPCRperformance. The points, from left to right represent dilutions offactors of 2.1, 3, 4, 5, 6, 7, 8, 10, 16 and 21. All results werenormalized to 1:5, as this was used in the protocol presented by ASDF.As can be seen by this figure, reaction inhibition is seen at low(1:2.1, 1:3) dilution factors. The total concentration of template, andhence the sensitivity of analysis is reduced with higher dilutionfactors. Thus, a dilution factor of 5× was used as the dilution factorin the final device. Error bars represent standard deviation of twosamples.

Example 1.7 Pre-Amplification Verification and Elution Efficiency

A device encompassing the design considerations outlined above wasdesigned, fabricated and tested in order to verify the combinedfunctioning of cell trapping, washing, and lysis, followed by reversetranscription and pre-amplification and to test sample elutionstrategies. A schematic for this device can be found in FIG. 7. Thisdevice featured the ability to capture 10 single cells in parallel, a 1nL cell processing chambers, 15 nL reverse transcription reactionchambers, 75 nL PCR pre-amplification reaction chambers, a factorialdemultiplexor, used to individually address each sample, and aperistaltic pump.

Ten single K562 cells were trapped and lysed. Human miRNA 16(hsa-mir-16) was then reverse transcribed, and relative copy numberdetermined via qPCR. The real-time PCR curves from this experiment canbe found in FIG. 8. All cells were expressing miRNA 16, and all curvescame up with an average cycle threshold of 26, and a standard deviationwithin 1 C_(t) of the mean.

Example 1.8 Elution Methods

As mentioned above, the design used to verify the combined functioningof the biochemical assays presented in FIG. 7 included a factorialdemultiplexor so that each chamber could be individually addressed, anda microfluidic peristaltic pump. Both of these design features wereintegrated in order to test elution methods. Because qPCR measuresrelative starting concentrations of DNA in solution, it is important toelute each chamber with the same volume. A variety of elution methodswere tested: 40 000 microfluidic peristaltic pump cycles,pressure-driven flow for a certain amount of time, elution onto Whatmanfilter paper followed by DNA resuspension in TE buffer, elution followedby sample drying and resuspension in a known volume and injecting fluidinto the device using a Harvard Apparatus syringe pump (PHD 2000). Theresults of each test are summarized in Table 1. All presented data isthe average of 10 replicates, and error is measured as the standarddeviation of the measurements.

TABLE 1 Summary of measurements to test elution methods. Elution MethodMean ± Standard Measurement Method Deviation Microfluidic Pump WeighPipette Tips  3.13 mg ± 0.32 (10%) BD 1 mL Plastic Syringe Weigh PipetteTips 4.10 mg ± 0.20 (5%) BD 1 mL Plastic Syringe qPCR on Cell 30.7 ±0.7   Lysate Cycle Whatman Filter Paper qPCR on Cell 23 ± 0.4  LysateCycle

While elution onto Whatman filter paper and elution followed by sampledrying produced the most reproducible results, these methods addedsignificant time to the workflow. It was determined that elution using asyringe pump and a high-precision glass syringe (Hamilton, 500 uL)produced both consistent elution results without significantly impedingthe experimental protocol. Because the syringe pump can be computercontrolled, this elution method is also fully automatable.

In order to assess the elution efficiency, the qPCR reaction presentedin the above section was brought to saturation (40 cycles, see FIG. 8)and the resulting product was eluted from the device by injecting 20 uLusing a syringe pump, thereby diluting the product 266×. The real-timecurves from these chambers can be seen in FIG. 9. Three of thesechambers were then eluted a second time in order to assess the elutionefficiency. On average, there was approximately an 813±5 fold decreasein relative abundance, corresponding to an elution efficiency of99.877%±0.0008%.

Example 1.9 Final Integrated Device Design

With all of the pieces required for highly multiplexed single cell geneanalysis in place, a device was designed that is capable of processingforty single cells, seven standards and an NTC all in parallel. Thedevice was designed to match the throughput of the Fluidigm 48.48Dynamic Array (DA) being used for endpoint quantification. A schematicof the device can be found in FIG. 10. This device features the abilityto process 40 single cells from up to four different populations (up tofour independent cell populations can be tested, each containing 10samples), seven standards, and a no template control, all in parallel.The inset shows an enlargement of one of the units in the array. Eachunit is connected to a reagent injection line a, and a cell loading linef. Control line b is used to isolate each unit from the reagentinjection line. Control lines c are used to individually address eachsample. Control lines d, e and g are used to isolate the cell processingregion from subsequent reaction chambers and neighboring processingunits. A reverse transcription reaction chamber is downstream of thecell processing region at h, immediately followed by a PCRpre-amplification chamber at j. Control line i is used to separate eachreaction chamber. Control line k is used to hydrate the PCR chamberduring thermocycling, and control line l is used to isolate the PCRreaction chamber. Pre-amplification product is eluted through the outletm. The scale bar is 1 cm, and the inset scale bar is 1 mm.

A basic schematic of the process is illustrated in FIG. 11. Briefly,cells are captured using the optimized capture weir described in Example1.1 (FIG. 11A). After the captured cells have been washed by flowingclean culture media over them (washing untrapped cells, debris and anyextracellular miRNA out of the device), valves then partition theloading channels into a linear array of 1 nL lysis chambers (FIG. 11B).Cells are then lysed by placing the entire device on a flatbedthermocycler, and heating it to 85° C. for seven minutes. After lysis,reverse transcription mix is injected through the reagent injectionlines, pushing the entire contents of each lysis chamber into thereverse transcription chamber (FIG. 11C). The reagent injection linesare flushed with water (FIG. 11D), and the device is then placed on aflatbed thermocycler, and a pulsed RT protocol is run. The RT product isthen pushed into the PCR pre-amplification chamber with PCR mix (FIG.11E). The device is left for one hour at room temperature to allow theRT product and PCR mix to diffusively mix, and the reagent injectionlines are once again washed, as depicted in FIG. 11D. The device is onceagain placed on a flatbed thermocycler, and a low-cyclepre-amplification PCR protocol is run. Following pre-amplification, acapillary pipette tip is plugged into each outlet port, and a syringe ina syringe pump is connected. Each sample is then serially eluted byinjecting 10 uL of elution mix into each chamber, diluting the product100× (FIG. 11F). After being eluted, each sample is then loaded into aseparate well on the Fluidigm 48.48 DA chip.

The entire process takes approximately 16 hours, and uses less than 5 μLof RT mix, 10 μL of pre-amplification mix, and 500 μL of qPCR mix. Intotal, there are approximately 210 pipetting steps. Performing anequivalent experiment in tubes would require more than 240 μL RT mix,960 μL of pre-amplification mix, and more than 46 nL of qPCR mix. Thistube-based experiment would require more than 4700 pipetting steps.Thus, the presented work results in an improved protocol, significantlyreducing the cost and time required to perform highly parallel, highlymultiplexed single cell analysis.

Example 2

An integrated microfluidic device that performs 300 parallel RT-qPCRassays and executes all steps of single-cell capture, lysis, reversetranscription, and qPCR is shown in FIG. 12A. To facilitate the precisecomparison of different samples and cell types, the device consists of 6independent sample-loading lanes, each containing 50 cell-processingunits for a total of 300 RT-qPCR reactions using approximately 20 μL ofreagents. Design elements were included to 1) allow for efficientdistribution of single cells without mechanical damage, 2) minimizebackground signal arising from free RNA or cell debris in the medium,and 3) avoid reaction inhibition by cell lysates in nL volumes.

In order to reduce device complexity and obviate the need for RNApurification, the device was optimized to be compatible withcommercially available assays that use “one-pot” RT-qPCR protocolsrequiring only the sequential addition of reagents into a singlereaction vessel. The rectangular box in FIG. 12A indicates the regiondepicted in FIG. 12B. Each cell-processing unit consists of a compoundchamber, formed by a cell capture chamber connected sequentially to twolarger auxiliary chambers for RT and qPCR (FIG. 12B). Each unit consistsof (1) a reagent injection line, (ii) a 0.6 nL cell capture chamber withintegrated cell traps, (iii) a 10 nL RT chamber, and (iv) a 50 nL PCRchamber (scale bar: 400 μm). FIG. 1C is an optical micrograph of twocell capture chambers with trapped single cells indicated by blackarrows. Each trap includes a cell funnel comprising deflectors to directcells into the capture region (scale bar: 400 μm). FIGS. 12D-I areschematic diagrams of the device's operation (scale bar for D-I: 400μm). In FIG. 12D, a single cell suspension is injected into the device.In FIG. 12E, cell traps isolate single cells from the fluid stream andpermit washing of cells to remove extracellular RNA. In FIG. 12F,actuation of pneumatic valves results in single cell isolation prior toheat lysis. In FIG. 12G, reagent for reverse transcription (RT) reaction(10 nL) is injected via a reagent injection line. In FIG. 12H, thereagent injection line is flushed with subsequent reagent for PCR. InFIG. 12I, reagent for qPCR is combined with RT product in a 50 nL qPCRchamber.

This simple fluidic architecture allows the implementation of eitherheat lysis followed by two-step RT-qPCR (FIG. 12D-I), or chemical lysisfollowed by one-step RT-qPCR. All lanes are connected to a common feedchannel which, following the completion of each reaction step, is usedto inject the next reaction master mix through the upstream chambers,thereby diluting the intermediate product (cell lysate or cDNA) andassembling the next reaction mixture. This parallelization of reactionassembly in a microfluidic format ensures equal timing of all reactionsteps and greatly reduces technical variability associated withpipetting and mixing steps in μL volumes. Fluorescence measurements wereperformed to ensure the efficient and reproducible transfer of reactantsat each step, showing that losses in sample transfer are below 5%. Tominimize device expense and complexity, temperature control andfluorescence detection were performed using peripheral hardwareincluding a CCD detector mounted above a flatbed thermocycler plate.

Example 2.1 Materials and Method Device Fabrication and Operation

Measurements of mRNA and miRNA abundance performed on single cells asdescribed above were found to consistent with average copy numbersmeasured by digital PCR on cell lysates (FIG. 20 c). Microfluidicdevices were fabricated by multilayer soft lithography (70, 71). Planarsilicon molds were defined by photolithography, using photomasksdesigned with CAD software (AutoCAD, Autodesk Inc.), and printed ontransparency films at a resolution of 20,000 dots per inch (CAD/Artservices). The ‘control’ mold was fabricated using SU8-2025 photoresist(Microchem, USA) to deposit valve features 24 μm in height. The ‘flow’mold was fabricated with three lithographic steps. First, the channelsfor reagent injection, and connections between chambers were fabricatedusing 13 μm high SPR220-7 photoresist (Shipley, USA). The SPR channelswere rounded to facilitate valve closure by incubation at 115° C. for 15minutes. A hard bake at 190° C. for 2 hours was used to prevent SPRphotoresist erosion during addition of subsequent layers. Second, thecell trap features were defined in 14 μm SU8-2010 photoresist(Microchem, USA). Finally, the large chambers and fluidic bus lines wereconstructed using 150 μm high SU8-100 photoresist. All photoresistprocessing was performed according to manufacturer specifications.

Microfluidic devices were cast from these molds in polydimethylsiloxane(PDMS, RTV615, General Electric, USA). Each device consists of a threelayer elastomeric structure with a blank bottom layer, a middle‘control’ layer with channels that act as valves by pushing up andpinching closed channels in the above ‘flow’ layer. The molds were firsttreated with chlorotrimethylsilane (TMCS, Aldrich) vapor for 2 min toprevent PDMS from bonding to the photoresist structures. The flow layerwas made by pouring a mixture of PDMS (5 parts RTV615A:1 part RTV615B)onto the flow mold, degassing, and then baking for 60 min at 80° C. Athin control layer was made by spin coating the control mold with PDMS(20 parts RTV615A:1 part RTV615B) at 1800 rpm and baking for 45 min at80° C. After baking, the PDMS of the flow layer was peeled from the flowmold and aligned to the control layer. Following a 60 min bake at 80°C., the bonded two layer structure was separated from the control mold,and channel access holes were punched. A blank layer (without channels)was prepared by spinning PDMS (20 parts RTV615A:1 part RTV615B) on ablank wafer (2000 rpm) and baking 45 min at 80° C. The bonded flow andcontrol structure was mounted on to the blank layer, and baked for 3hours at 80° C. Finally, the three layer bonded structure was removedfrom the blank mold, diced into individual devices, and these were eachbonded to clean glass slides by baking overnight at 80° C.

The device operation requires control of 9 pneumatic valves and may beoperated using a simple manifold of manual valves. For the current studya semi-automated implementation was used in which microfluidic valveswere controlled by solenoid actuators (Fluidigm Corp., San Francisco)controlled through a digital input output card (NI-DAQ, DAQ-32H,National Instruments) operated using LabView drivers (NationalInstruments). Tygon tubing connected the solenoids to the microfluidicdevice by 20 gauge stainless steel pins (Small Parts Inc.) fitted intothe control line ports. Krytox (DuPont) oil was used as the fluid in thecontrol lines, and the valves were actuated with 30 psi pressure.

Microfluidic Single Cell RT-qPCR

The device was designed to be compatible with commercially availableRT-qPCR products. A protocol for heat lysis, followed by a 2-stepRT-qPCR was used with miRNA and OCT4 mRNA assays. Alternatively, achemical lysis, followed by 1-step RT-qPCR was used for mRNAmeasurements of SNVs and GAPDH.

Single Cell Transcript Measurements by Heat Lysis and 2-Step RT-qPCR

The device was primed by flowing PBS containing 0.5 mg/mL bovine serumalbumin (BSA) and 0.5 U/μL RNase Inhibitor through all channels, whilekeeping the RT, and PCR chambers empty and isolated by valves. The BSAhelped prevent cells from adhering to channel walls. After priming, butprior to cell loading, all valves were closed. A single cell suspensionwas injected into the device by applying pressure (˜2-3 psi) tomicrocapillery pipette tips plugged into the sample inlets. The sampleinlets were first dead-end filled against an inlet valve to prevent airbubbles from entering the device. The sample inlet valves, cell chambervalves and outlet valve were opened to allow the cell suspension to flowthrough the sample channels. Cells were loaded into the device suspendedin culture media (directly from culture). Cell loading concentrationswere kept between 5×10⁵ cells/mL and 1×10⁶ cells/mL, resulting in over80% occupancy of cell traps with single cells in 1-2 min at a flow rateof approximately 20 nL/s. Lower concentrations were found to requireproportionately longer times to achieve high occupancy of trapped singlecells. Concentrations greater than 2×10⁶ cells/mL were found tooccasionally clog the inlet port or the channel at trap locations. Aperistaltic pump was integrated into the device for controlling the flowrate, however pressure driven flow was used for the current study.

After injecting the cell suspension and trapping single cells the cellsample inlet valve was closed, and the cells were washed by flushing theline with the PBS solution used to prime the device. This removeduntrapped single cells, extracellular RNA, and debris. Following on-chipwashing, the cell chamber valves were closed to partition the cellloading channel and isolate individual cell reactors. Visual inspectionof the cell capture chambers with a microscope was used to confirm andcount the number of cells in each chamber. The cells were lysed byplacing the microfluidic device onto a flatbed thermocycler and heatingto 85° C. for 7 minutes (and then cooled to 4° C.).

Reverse transcription (RT) was performed in the device by using the ABIHigh Capacity Reverse Transcription kit (68), with the addition of asurfactant to prevent adsorption of nucleic acids and proteins to PDMSsurfaces (2 μL 10× Reverse Transcription Buffer, 4 μL 5×RT stem-loopmiRNA primer from ABI, 1 μL 100 mM dNTPs, 1.34 μL of 50 U/μL MultiscribeReverse Transcriptase, 0.26 μL of 20 U/μL RNase Inhibitor, 2 μL 1% Tween20, 9.4 μL PCR grade water). The RT mix was loaded into the device, andflushed through the reagent injection channels. RT reagent was injectedinto the reaction by opening the valve connecting the cell chamber tothe RT chamber, and the valve connecting the cell chamber to the reagentinjection line. The RT chamber was dead-end filled before closing theconnection to the reagent injection line. A pulsed temperature RTprotocol was carried out by placing the microfluidic device on a flatbedthermocycler (2 min at 16° C., followed by 60 cycles of 30 seconds at20° C., 30 seconds at 42° C., and 1 second at 50° C.). RT enzyme wasinactivated at 85° C. (5 min), and then the device was cooled to 4° C.

The PCR reagent was prepared with 25 μL of 2× TaqMan Universal MasterMix (ABI), 2.5 μL 20× Real-Time miRNA assays (primers and probe, ABI), 5μL of 1% Tween 20, and 7.5 μL of PCR grade water. The PCR reagent wasflowed through the reagent injection channels to flush away the RTreagent. Valves were opened and the PCR reagent was injected to dilutethe RT product into the PCR reaction chamber. After completely fillingthe PCR reaction chamber, the valves closing the PCR chambers wereactuated, and the device was transferred to an enclosure for real-timePCR (Prototype version of Biomark™ Instrument, Fluidigm CA). Thereal-time PCR enclosure consists of a custom flatbed thermocycler, axenon arc lamp and filter set, and a charged coupled device (CCD) imagerwith optics for fluorescent imaging of the entire device periodicallyduring PCR thermocycling (see description of real-time PCRinstrumentation below). PCRs were thermocycled with the followingconditions: 10 min at 95° C., followed by 50 cycles of 15 s at 95° C.and 1 min at 60° C. Images were acquired at 60° C.

Single Cell Transcript Measurements by Chemical Lysis and 1-Step RT-qPCR

Measurements of mRNA transcripts (SP1, GAPDH) were performed using theCells Direct kit (Invitrogen, USA). Operation of the microfluidic devicefor chemical lysis and 1-step RT-qPCR was similar to the methodsdescribed for heat lysis and 2-step RT-qPCR with several distinctions.The device was primed and cells were washed with PBS containing 0.5mg/mL BSA. Additional RNase Inhibitor was omitted as the chemical lysisbuffer (10 μL lysis resuspension buffer, 1 μL lysis enhancer solution,Invitrogen, USA) contained RNA stabilizing agents. Cell loading was thesame as in the heat lysis and 2-step RT-qPCR scenario. Single cells werelysed by injecting a chemical lysis buffer through the cell capturechamber and filling the 10 nL chamber (used for RT reagent injection inthe 2-step protocol). The lysis reaction was incubated at roomtemperature for 10 minutes, followed by heat inactivation of the lysisreagent by placing the device on a flatbed thermocycler and incubatingat 70° C. for 10 minutes. The one-step RT-qPCR mix (1 μL of SuperScriptIII RT/Platinum Taq Mix, 25 μL of 2× Reaction Mix (with ROX referencedye), 2.5 μL of 20× Taqman Assay (primers and probes, ABI), 1 μL of 50mM MgSO₄, 5.5 μL of H₂O, and 5 μL of 1% Tween 20) was then combined withthe cell lysate into the final 50 nL reaction chamber. The device wastransferred to the real-time PCR enclosure for temperature control andimaging of the 1-step RT-qPCR (20 min at 50° C. for RT, followed by ahot-start at 95° C. for 2 min, and 50 cycles of 15 s at 95° C. and 30 sat 60° C.).

Digital PCR Experiments

For mRNA digital PCR analysis cells were washed with PBS containing 0.5mg/mL BSA, lysed in chemical lysis buffer, reverse transcription wasperformed in tubes according to the protocol described above, and theresulting cDNA product was loaded into digital PCR arrays. For miRNAstudies, cells were lysed in PBS containing 0.5 mg/mL BSA and 0.5 U/μLRNase inhibitor. Reverse transcription was performed using miRNAstem-loop primers (Applied Biosystems, USA) and the High Capacity cDNAReverse Transcription kit (Applied Biosystems, USA) in 10 μL volumes.Prior to injection into microfluidic digital PCR arrays, RT product wasadded to the PCR reagent as in the on-chip 2-step RT-qPCR protocoldescribed above. Thermal cycling of digital PCR arrays was alsoperformed using the same protocols as described above. PDMS digital PCRarrays consisting of 765 2 nL individual PCR chambers, of similar designto those described in Warren et al. (72), were fabricated by multilayersoft lithography. After thermal cycling, positive chambers were countedand actual molecule numbers were derived based on the binomialdistribution.

System for Real-Time PCR

The BioMark™ Reader is a commercially available real-time PCR instrumentdeveloped by Fluidigm and designed to run Fluidigm Integrated FluidicCircuits (IFCs). The prototype version of this system allowed access tothe flatbed thermocycler inside the enclosure, permitting the use ofcustom microfluidic devices in addition to the intended commercial IFCs.

Fundamental specifications for data collection include:

-   -   Image Resolution and bit depth: 4 Megapixel, 16 bit    -   Filters: FAM: Ex 485/20 Em 525/25;        -   VIC: Ex 530/20 Em 570/30;        -   ROX: Ex 580/25 Em 610/15;        -   QAS: Ex 580/25 Em 680/25,    -   Light Source: 175 W xenon arc bulb

RT-qPCR Assays

Measuring mRNA in the presence of genomic DNA requires primers designedto specifically target mature mRNA sequences. In many cases, this can beaccomplished by designing intron-spanning primers. A specially designedstem-loop RT primer system (Applied Biosystems) is used for the specifictargeting of mature miRNAs.

TaqMan assays for GAPDH (Applied Biosystems, Assay ID Hs99999905_m1) andmiRNAs were obtained from Applied Biosystems. For GAPDH, a controlexperiment omitting the reverse transcriptase was performed off-chip, inmicroliter volumes with bulk cell lysate (at equivalent concentration ofa single cell on-chip, 10⁵ cells/mL), and showed no amplification after40 cycles of PCR.

OCT4 (POU5F1) primer sequences were obtained from RTPrimerDB¹ andsynthesized by Biosearch Technologies Inc; Forward primer: ACC CAC ACTGCA GCA GAT CA, Reverse primer: CAC ACT CGG ACC ACA TCC TTC T, Probe:Quasar⁶⁷⁰-CCA CAT CGC CCA GCA GCT TGG-BHQ-2, RT primer: TTG TGC ATA GTCGCT GCT TGA T. Measurement of OCT4 in single hESCs by microfluidicRT-qPCR without reverse transcriptase showed no amplification after 40cycles of PCR.

BHQ-Plus probes with enhanced duplex stabilization (BiosearchTechnologies Inc) were used for SNV detection to allow for shortersequence lengths and increased specificity. The SNV location for the SP1locus was selected from Table 2 in Shah et al. (73). Two hundred byflanking this location on the hg18 sequence were used for assay designusing Primer3. The resulting primer and probe sequences are as follows(the SNV is underlined):

SP1 Mutant Probe: FAM-AGGCCAGCAAAAACAAGG-BHQ-15′ Modification: FAM, 3′ Modification: BHQ-1 Plus. Tm=62.7° C.SP1 WT probe: Cal Fluor-CAGGCCAGCAAAAAGAA-BHQ-15′ Modification: CAL Fluor Orange 560, 3′ Modification: BHQ-1 plus.Tm=62.1° C.

SP1 Forward Primer: CCAGACATCTGGAGGCTCATTG Tm=65.8° C. SP1 ReversePrimer: TGAACTAGCTGAGGCTGGATA Tm=66.0° C.

Control experiments without reverse transcriptase showed positiveamplification. Therefore the measurement of SP1 mutant and wilde-typeabundance in single cells by RT-qPCR does not discriminate betweenmature mRNA transcript and genomic DNA.

Image Analysis

Fluorescence images of the entire device were taken in at least twodifferent colors (one passive reference dye and one or more reporterdyes) after each PCR cycle and were analyzed using custom scriptswritten in MATLAB (MathWorks) to generate real-time amplificationcurves. Reaction chambers were segmented from the rest of the imageusing the first image of the passive reference dye. The image wasmanually rotated so that all of the reaction chambers were square withthe edges of the image. Next, the average image intensities across eachrow and column were calculated and a threshold was manually set todifferentiate bright areas from background. Regions containing bothbright rows and bright columns were assigned to the reaction chambers.

All subsequent images were automatically aligned to this initial imageby minimizing the absolute distance between the average row and columnintensities of the initial image, and the one being analyzed. For eachimage, the intensities of the reporter and passive dyes were recordedfor each reaction chamber. Real time amplification curves were generatedby normalizing the intensity of each reporter dye to that of the passivedye. Linear components were removed from these curves by fitting theequation of a line to the pre-exponential region and extrapolating andsubtracting the result from the entire curve. The threshold fordetermining CT values was automatically determined as the mediannormalized fluorescence value at the maximum second derivative of allamplification curves.

mRNA Fish

Microfluidic measurements of Oct4 expression in single cells were foundto be consistent with OCT4 measurements obtained by mRNA FISH. For mRNAFISH measurements, cells grown on LABTEK chambered cover-glass werewashed with PBS, fixed in 4% formaldehyde for 10 min at room temperatureand permeabilized in 70% EtOH at 40° C. overnight. The next day cellswere rinsed with wash buffer (15% Formamide in 2×SSC) and thenhybridized with the appropriate dilution of mRNA FISH probes specific toOCT4 (see Table 2) in hybridization solution (dextran sulfate, YeasttRNA, NEB, BSA, 15% Formamide in 2×SSC) overnight at 30° C. The nextmorning the OCT4 hybridization solution was aspirated and cells weresequentially rinsed and incubated with wash buffer at 30° C. for 30minutes then washed with 2×SSC. One drop (25 μL) of Slowfade GOLDantifade reagent with DAPI was the added to the cells, coveredimmediately with a cover slip, and imaged. Stacks of 32-64 mRNAhybridization images (spaced by 0.5 μm) were acquired for each cellusing a Leica DMI 6000B inverted microscope with a 100× objective (N.A.1.3) in DAPI and Texas Red filter spectra.

Fluorescent spots corresponding to individual mRNA molecules in eachimage stack were evaluated manually since automatic thresholding usingpreviously reported algorithms were found to be unreliable. Difficultyin automating this process was attributed to inconsistent signal tonoise using reported protocols and may be related to the thickness ofhESC cells (˜15 μm). In addition, manual intervention was needed toascertain the boundaries of adjacent cells. To optimize the signal tonoise we systematically varied the probe concentration, incubation time,incubation temperature as well as the formamide concentration in thehybridization buffer solution.

TABLE 2 Sequence of mRNA FISH probes specific to OCT4 SEQ Probe % IDProbe (5′->3′) Name # Position GC NO: tgaaatgagggcttgcgaag OCT4_1 1 2 501 aaatccgaagccaggtgtcc OCT4_2 2 61 55 2 atcacctccaccacctggag OCT4_3 3 9560 3 aggtccgaggatcaacccag OCT4_4 4 138 60 4 aggagggccttggaagctta OCT4_55 161 55 5 aatcccccacacctcagagc OCT4_6 6 215 60 6 atccccccacagaactcataOCT4_7 7 253 50 7 actagccccactccaacctg OCT4_8 8 289 60 8tcaggctgagaggtctccaa OCT4_9 9 322 55 9 agttgctctccaccccgact OCT4_10 10354 60 10 ttctccttctccagcttcac OCT4_11 11 418 50 11 ctcctccgggttttgctccaOCT4_12 12 440 60 12 ttctgcagagctttgatgtc OCT4_13 13 466 45 13cttggcaaattgctcgagtt OCT4_14 14 488 45 14 tgatcctcttctgcttcagg OCT4_1515 510 50 15 atcggcctgtgtatatccca OCT4_16 16 533 50 16aaatagaacccccagggtga OCT4_17 17 560 50 17 tcgtttggctgaataccttc OCT4_1818 582 45 18 taagctgcagagcctcaaag OCT4_19 19 612 50 19gcagcttacacatgttcttg OCT4_20 20 636 45 20 tccacccacttctgcagcaa OCT4_2121 661 55 21 gattttcattgttgtcagct OCT4_22 22 684 35 22tctgctttgcatatctcctg OCT4_23 23 706 45 23 actggttcgctttctctttc OCT4_2424 743 45 24 ttgcctctcactcggttctc OCT4_25 25 766 55 25ctgcaggaacaaattctcca OCT4_26 26 788 45 26 atctgctgcagtgtgggttt OCT4_2727 814 50 27 atccttctcgagcccaagct OCT4_28 28 851 55 28ttacagaaccacactcggac OCT4_29 29 874 50 29 tagtcgctgcttgatcgctt OCT4_3030 910 50 30 ctcaaaatcctctcgttgtg OCT4_31 31 932 45 31ctgagaaaggagacccagca OCT4_32 32 954 55 32 agaggaaaggacactggtcc OCT4_3333 076 55 33 atagcctggggtaccaaaat OCT4_34 34 1010 45 34agtacagtgcagtgaagtga OCT4_35 35 1038 45 35 ttccccctcagggaaaggga OCT4_3636 1064 60 36 tgacggagacagggggaaag OCT4_37 37 1086 60 37agtttgaatgcatgggagag OCT4_38 38 1116 45 38 attcctagaagggcaggcac OCT4_3939 1139 55 39 ttttctttccctagctcctc OCT4_40 40 1176 45 40aaaaaccctggcacaaactc OCT4_41 41 1200 45 41 ccttagtgaatgaagaactt OCT4_4242 1226 35 42 accctttgtgttcccaattc OCT4_43 43 1249 45 43aaccagttgccccaaactcc OCT4_44 44 1278 55 44 cattgaacttcaccttccct OCT4_4545 1300 45 45 gtgggattaaaatcaagagc OCT4_46 46 1322 40 46ccaggcttctttatttaaga OCT4_47 47 1359 35 47 aagtgtgtctatctactgtg OCT4_4848 1381 40 48

Cell Culture

K562 cells were cultured in Dulbeco's Modified Eagle Medium (DMEM)(Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco). PurifiedRNA was extracted from K562 cells using RNA MiniPrep (Qiagen, USA).

CA1S hESCs (74, 75) were propagated in mTeSR (76) basal medium (STEMCELLTechnologies, Inc., Vancouver, BC, Canada), additionally supplementedwith antibiotic-antimycotic (100 U/mL penicillin, 100 mg/mL streptomycinand 0.25 mg/mL amphotericin B) (Invitrogen, Carlsbad, Calif., USA). Uponpassaging, hESCs were washed with phosphate-buffered saline (PBS) priorto incubating with TrypLE Express (Invitrogen, Carlsbad, Calif., USA) at37° C. for 10 minutes to detach single hESCs from 4-8 day-old culturesdepending on confluency. TrypLE Express was neutralized with mTeSRsupplemented with antibiotic-antimycotic and suspensions were thentransferred into new tissue culture dishes containing a precoated layerof 1:30 diluted Matrigel (Becton Dickinson, San Jose, Calif., USA) andmTeSR supplemented with antibiotic-antimycotic. For differentiation,mTeSR was replaced with Dulbecco's modified eagle medium with 10% fetalbovine serum (FBS) 1 day after plating cells.

When harvesting hESCs for qRT-PCR, cells were incubated with TrypLEExpress (Invitrogen, Carlsbad, Calif., USA) at 37° C. for 20 minutes inorder to produce a more uniform single cell suspension from 4-8 day-oldcultures.

Cryo-vials of primary cells isolated from a lobular breast cancermetastasis were provided by the BC Cancer Agency in accordance withethical guidelines of the University of British Columbia. To increaseviability, cells were transferred to fresh culture medium and incubatedfor 2 days before analyzing in the microfluidic device.

Transfer Efficiency Measurements

A solution containing 10 μM FAM-labeled 40-mer poly-A oligonucleotides(IDT, USA), 0.1% Tween 20, and ROX passive reference dye (fromCellsDirect kit, Invitrogen, P/N 54880) diluted 100× was loaded into thecell capture chambers and sequentially pushed into the 10 nL and 50 nLchambers with water containing 0.1% Tween 20, and ROX reference dyediluted 100×. Fluorescence images acquired of FAM and ROX were used tomeasure the transfer of oligonucleotides from one chamber to the next.The transfer efficiency for each chamber was calculated as (InitialSignal−Final Signal)/(Initial Signal), where Signal=(FAM Intensity−FAMBackground)/(ROX Intensity−ROX Background). A conservative estimate ofthe lower bound of transfer efficiency was taken to be one standarddeviation from the mean measurement of transfer efficiency.

Cell Capture Measurements

A custom microfluidic device with a linear array of cell trap geometrieswas fabricated using protocols described above. The device was mountedon an inverted microscope (Leica DM IRE2) and imaged in bright fieldusing a CCD camera (Hamamatsu ORCA-ER). The device was primed with 0.05%bovine serum albumen (BSA) (Gibco) in phosphate-buffered saline (PBS)(Gibco). Prior to loading in the device, cells were washed twice infresh culture media (Dulbeco's Modified Eagle Medium (DMEM) (Gibco)supplemented with 10% Fetal Bovine Serum (Gibco)). After the final washcells were resuspended to be at a concentration of 1 million per mL.Input sample viability was measured with the Cedex Automated CellCounter (Roche innovatis AG).

To measure the capture efficiency, cells were pumped through the arrayusing a downstream microfluidic peristaltic pump at a rate ofapproximately 1 nL/second and the number of cells that bypassed eachtrap before a successful trapping event was recorded. These counts werefit using a maximum-likelihood estimator for a geometric distributionwith the fitdistr function (MASS package version 7.3-6) in R (version2.11.1). Efficiencies are reported as the probability of a successfulcapture for each cell.

To measure cell viability after loading, cells were loaded into thearray using pressure driven flow as described above until high trapoccupancy was observed. 0.2% Trypan Blue (Gibco) in PBS was then flowedover the trapped cells. Viability was calculated as the number ofunstained cells divided by the total number of cells.

Cell diameter was measured from Cedex images and images of cells trappedin the microfluidic device using ImageJ (version 1.43u). A two samplet-test was used to test the hypothesis that the resulting sizedistributions were significantly different. The assumption of equalvariance was tested using an F test. For optimized cell trap geometriesthe cell trapping efficiency was improved to 87% by bringing the cupwithin one cell diameter of the focuser and by including a small bypassshunt through the cup, similar to the cup geometry presented in Skelleyet al. (10).

Mixing by Diffusion

Mixing of solutions by diffusion was characterized in the microfluidicdevice by loading fluorescently labeled 40 bp poly-A oligonucleotidesinto the 10 nL chambers, and pushing the contents of the chamber intothe adjacent 50 nL chambers. Time-lapse imaging was used to measure theevolution of the distribution of fluorescently labeled oligonuceotidesin the PCR chambers over time (Figure S7). The standard deviation of thepixel intensities in each chamber through time was used as a metric ofmixing. The resulting curves of all analyzed chambers (N=200) were eachfit to a decaying exponential using least squares regression todetermine the characteristic mixing time constant. This resulted in amean mixing time of 15.2±1 minutes.

Using the Stokes-Einstein relation and assuming a random coil weestimate the diffusion constant of a 40 by oligonucleotide to be:

$D = {\frac{K_{B}T}{6{\pi\eta}\; R} \cong \frac{K_{B}T}{6{{\pi\eta}\left( {Nd}^{2} \right)}^{1/2}}}$

, where K_(B)T is the thermal energy (4.1 pN·nm), η is the fluidviscosity (˜0.001 kg/m·s), d is the length of a DNA base pair (˜3.3Angstroms), and N is the number of base pairs. This yields a value ofapproximately 1.15×10⁻¹⁰ m²s⁻¹, which is comparable to the diffusionconstant of polymerase, the largest molecule in the PCR mix. Since thetemplate solution constitutes only ⅕ of the final PCR reaction it mustdiffuse the longest distance to equilibrate across the chamber.Therefore, the measured diffusion time of 15.2 minutes represents anupper bound to the time constant for complete mixing of all components.

Example 2.2 Precision and Sensitivity of Microfluidic RT-qPCR

Chamber volumes were designed to ensure sufficient dilution between eachprocessing step to avoid reaction inhibition while at the same timemaintaining high template concentrations and assay sensitivity.

FIG. 13A is a fluorescence image of the entire device showing 300reactions in 6 lanes, taken after 40 cycles of PCR from dilution seriesof purified total RNA from K562 cells. From left to right the samplesare 40 pg/chamber, 5 pg/chamber, 625 fg/chamber, 78 fg/chamber, 10fg/chamber, and no-template control (NTC). Single molecule amplificationat limiting dilution results in a digital amplification pattern for 10fg and 78 fg lanes. No amplification is observed in NTC lane (N=50).FIG. 13B shows 300 real time amplification curves generated fromprocessing sequences of images similar to FIG. 13A. The threshold fordetermining CT values is indicated by the dashed line. FIG. 13C depictson-chip (lower) and off-chip (upper) RT-qPCR for GAPDH from a 8× serialdilution of purified total RNA shows improved sensitivity in nL volumereactions. In the microfluidic system, CT values for the 10 fg samplecorrespond to single molecule amplifications detected in 19 of 50chambers. The mean and standard deviation from single cell measurementsis shown in green for both on and off-chip analysis. CT values obtainedon chip correspond to a mean of 20 pg of RNA per cell. Off-chipmeasurements of single K562 cells washed twice in PBS and isolated byglass capillary exhibit artificially increased levels due to residualsignal from debris and free RNA in the supernatant. Cells weretransferred in approximately 2 μL of supernatant, which was measured tocontain ˜20 pg of extracellular RNA. Error bars represent standarddeviation of measured CT values for all amplified reactions. FIG. 13Dshows the real-time amplification curves of GAPDH in K562 cell lysatedilutions. FIG. 13D shows the measured CT values for GAPDH in dilutionseries of cell lysate. No inhibition occurs for single cell lysates.

Cell lysate dilutions showed that reaction inhibition becomessignificant at concentrations in excess of 0.2 cells/nL, or 10 cells per50 nL reaction (FIG. 13D). Experiments in tubes were performed todetermine that a dilution ratio of at least 5:1 (PCR mix:RT product) isoptimum for PCR efficiency. We therefore designed our combined reactorsto have an aggregate total volume of 60.6 nL, consisting of a 0.6 nLcell capture chamber, a 10 nL RT chamber, and a 50 nL qPCR chamber.These volumes allow for the reliable amplification of single molecules(FIG. 13A), and result in a final template concentration of 330 ng/mLwhen starting from a single cell equivalent of RNA (20 pg). The use oflarger volume RT and PCR chambers has the added advantage of reducingtheir surface to volume ratio, thereby minimizing reagent evaporationthrough the gas permeable device material (polydimethylsiloxane).

The sensitivity and precision of RT-qPCR in the device was tested byperforming measurements of GAPDH expression over an 8-fold dilutionseries of total RNA, ranging from 40 pg (˜2 cell equivalents) to 10 fg(˜ 1/2000 cell equivalents). RNA was purified from K562 cells, a BCR-ABLpositive human cell line derived from a patient with chronic myeloidleukemia (77) (FIG. 13A-C). The efficiency of amplification wasdetermined over the four highest template concentrations (40 pg, 5 pg,625 fg, 78.125 fg) as the slope from a linear least squares fit of log2(C) vs. CT, and was found to be 0.988±0.055. The standard deviation ofCT values was less than 0.15 at the three highest concentrations(s.d.=0.08, 0.10, 0.14 for the 40 pg, 5 pg, and 625 fg samplesrespectively), indicating uniform amplification across the array andtechnical error of less than 10% in absolute concentration, near thelimit of qPCR precision. The highest measurement variability wasobserved in the 78 fg sample, where shot noise (Poisson sampling noise)is most pronounced and accounts for approximately 50% of the measurementvariance. Template amounts below 625 fg resulted in a digital patterncharacteristic of single molecule amplification (49/50 for 78 fg, 19/50for 10 fg) and consistent with the expected occupancy of chambers asdetermined by a binomial distribution (2).

Based on the frequency of single molecule detection in the 10 fg sample,we measured the average copy number of GAPDH to be 979±240 transcriptcopies per single cell equivalent (20 pg) (FIG. 13). A comparison of CTvalues obtained from on-chip qPCR from cDNA synthesized off-chipdemonstrated that on-chip RT efficiency is equal to that obtainedoff-chip when working from the same RNA concentrations. FIG. 14 shows acomparison of GAPDH measurements from K562 cell lysate with RT performedin the microfluidic device or RT performed in tubes prior to qPCR in thedevice. Obtained CT values (inset) show no significant difference inefficiency. Finally, comparison of the same dilution series of RNA,assayed for GAPDH both on-chip and in tubes (20 μL volume) (FIG. 13C),showed that on-chip analysis provides improved sensitivity.

Example 2.3 Cell Capture

Cell capture chambers incorporated funnels (deflectors) located 22.5 μmfrom the trap, to focus cells into the central streamlines where captureis most efficient (FIG. 12C). Using these structures a high single celloccupancy of array locations was achieved (FIG. 15A, B). Over 8 separateexperiments, a loading protocol of ˜60 seconds (106 cells/mL, 20 nL/sper lane) resulted in the successful isolation of single cells in1518/1700 chambers (89.3%), with a cell capture efficiency of 5.0±0.5%.Staining with Trypan Blue™ was used to assess the viability of cellsafter loading and was determined to be equivalent to the viability ofthe input sample (97.4% viability vs. input 96.8%). Finally,measurements of the distribution of cell diameters prior to and afterloading indicated that cell trapping did not introduce significant bias(p=0.67, two sample t-test) in selecting cells of different sizes FIG.16 provides histograms showing the size distribution of cells inoriginal sample as measured by Cedex (FIG. 16A) are consistent with thesize distribution of cells isolated by microfluidic traps (FIG. 16B),with the assumption of spherical cell shape the distribution ofdiameters of trapped cells corresponds to a mean volume of 4.2 pL with astandard deviation of 2.0 pL. This cell trap geometry and loadingprotocol were used in all subsequent qPCR measurements presented below.Further improvement of trap and funnel (i.e deflector) geometries werefound to achieve fill factors of >99% (100 single cells captured out of100 traps analyzed) and cell capture efficiencies of 87.0±4.5%, withcell viability again matching the input sample (>98%) and notsignificantly biasing cell sizes (p=0.35, two-sample t-test), makingthis method applicable to the analysis of limited quantity samples suchas rare stem cells or clinical samples.

FIG. 15A shows the locations of cells in each chamber along all 6 lanesof a device, as determined by brightfield microscopy, are represented aswhite circles and overlaid on a heat map of CT values obtained fromRT-qPCR measurements of GAPDH in K562 cells. Dark circles indicate NTC.FIG. 15B is a scatter plot showing CT measurements for experiment shownin FIG. 15A. The histogram on the inset shows 93.2% single celloccupancy. FIG. 15C shows the distribution of the number of GAPDHtranscripts measured in single K562 cells (N=233).

Example 2.4

The immobilization of cells in traps in cell capture chambers was alsoused for on-chip washing of cells prior to lysis to remove free RNA,cellular debris, and untrapped cells that would otherwise give rise tobackground signal or result in low single cell occupancy. FIG. 17Aprovides measurements of GAPDH in cells washed in PBS off-chip prior toinjection into microfluidic device, without an on-chip wash containbackground signal from template in supernatant. Without on-chip washing,untrapped cells remain in the capture chambers, resulting in fewersingle cell measurements (histogram inlayed). As shown in FIG. 17B,on-chip washing was found to reduce the background signal from free RNAin the supernatant, and dramatically increased the number of singlecells analyzed. As seen in FIG. 18, detection of residual RNA afterwashing is dramatically reduced by comparison to off-chip results due tosmall volume processing. FIG. 18A depicts the locations of cells in eachchamber along all 6 lanes of a device, as determined by brightfieldmicroscopy, and represented as white circles and overlaid on a heat mapof CT values obtained from RT-qPCR measurements of miR27a in K562 cells.Shaded circles indicate NTC. FIG. 18B is a fluorescence image of theentire device corresponding to experiment depicted in FIG. 18A after 30PCR cycles. Cell corpses remain after heat lysis and are visible aspunctuate fluorescent spots adjacent to reaction chambers.

FIG. 17C provides a comparison of GAPDH measurements from loadingpurified RNA and washing, or not washing, the cell capture chambers. Theefficiency of chamber washing, determined by loading purified RNAtemplate (36.5 ng/mL), followed by washing and RT-qPCR analysis,was >99.99% (1.1′104 copies measured without wash, 0 copies detectedafter washing). In addition, RT-qPCR measurements testing different cellloading and washing protocols demonstrated that on-chip washing allowsfor loading directly from culture medium with low background as comparedto off-chip wash steps followed by analysis in μL volumes (FIG. 17C).Importantly, on-chip washing allows for lysis within seconds of washing,thereby minimizing spurious transcriptional responses that may arisefrom sequential medium exchange and spin steps.

Example 2.4

The efficiency and reliability of on-chip cell processing was evaluatedby comparing the GAPDH measurements of purified RNA to measurementsperformed directly from single K562 cells (FIG. 13C, FIG. 15C). K562cells were loaded directly from culture medium followed by washing andanalysis using a chemical lysis and one-step RT-qPCR protocol (CellsDirect™, Invitrogen). Using a CT threshold of 31.5, corresponding to themean CT of a single molecule of GAPDH(CT=30.5) plus two standarddeviations (s.d.=0.5), successful amplification was observed in 100% ofsingle cells (N=233) (FIG. 15A-B). Adjacent chambers that did notcontain a cell were distinguishable from single cell measurements withan average delta CT value of 5.7 (5 empty chambers, 3 of whichamplified) (FIG. 15A-B). A log-normal distribution of GAPDH in singlecells was observed with mean CT values of 20.3 (s.d.=0.8) and an averageof 1761 (s.d.=648) copies per cell (FIG. 15C). Additionally, the mean CTof 20.3 observed for single cells matches measurements of single cellequivalent lysate (CT=20.2, FIG. 13D). Using digital PCR on cDNAprepared from K562 cell lysate, an average of 1229±72 GAPDH moleculesper single cell equivalent was measured. Finally, as expected, RT-qPCRmeasurements from chambers loaded with more than one cell show reducedvariability and lower CT values (FIG. 16A, FIG. 19). AS shown in FIG.19, measurement of miR16 in hESC cell aggregates demonstrates that thenumber of cells is reflected in corresponding cycle threshold (CT)values.

Example 2.5 Application to Measurement of Single Cell miRNA Expression

The technology was next applied to the study of single cell miRNAexpression. The short length of miRNAs (˜22 nucleotides) makes themdifficult to detect by hybridization approaches, so that RT-qPCR is thedominant quantification strategy. To demonstrate the robustness andthroughput of this technology, a total of 1672 single cell measurementswere performed to examine single-cell variability in the expression of 9miRNAs spanning a wide range of abundance (>16000 copies per cell to<0.2 average copies per cell). K562 cells were again chosen as aheterogeneous population for this study since they are known to exhibitmixed characteristics of erythrocytes, granulocytes, and monocytes (77,78). The expression of miR-16, a highly expressed microRNA that is foundin many tissue types (79), was evaluated as an internal standard fornormalization (57). miR-16 was log-normally distributed across K562cells, but with slightly lower expression and notably tighter regulationthan GAPDH, having an average of 804 (s.d.=261) copies per cell and astandard deviation of 30% (mean CT=21.4, s.d.=0.4). Matched experimentson single cells, isolated by micropipette into 20 μL volume tubes,displayed an increase in measurement variability to ˜90% (mean CT=29.5,s.d.=0.9), demonstrating the improved precision of parallel microfluidiccell processing in nL volumes (FIG. 20A). The observed shift in mean CTvalues between on and off-chip measurements is due to lower templateconcentrations, and hence increased required PCR cycles, in the off-chipsamples.

(C) (D) 2072 single cell measurements of the expression of 9 miRNA inK562 cells. Reflected histograms represent the expression distributionsfor each miRNA.

To demonstrate the utility of this device for measuring differentialexpression in single cells, the expression of miR-223, a miRNAimplicated in myeloid differentiation, was measured. In contrast tomiR-16, K562 cell miR-223 expression was found to be highly variable(mean CT=22.2, s.d.=1.6, copy number=513, s.d.=406) and was notlog-normally distributed (FIG. 20B; the right-most bar indicates cellsfor which miR-223 was not detected (ND)), consistent with the knownfunctional heterogeneity of K562 cells. These measurements highlight theutility of single cell miRNA expression analysis for assessing theheterogeneity of cell populations and for identifying miRNAs that areuseful biomarkers of cellular state. To further explore thispossibility, the expression of an additional 7 miRNAs (9 total) wasmeasured, and the patterns of single cell expression in K562 populationswere plotted. FIG. 20C shows mean single cell miRNA copy numbersmeasured by RT-qPCR in the microfluidic device compared to digital PCRmeasurements from bulk cell lysate. Error bars represent standarddeviation of single cell measurements for each miRNA. Following theprocedure described above, single molecule CT values obtained by digitalPCR were used to translate measured CT values to absolute copy number.Assuming 100% efficient amplification, the copy number, calculated as2(CT (single cell)−CT (single molecule)), was observed to correlate well(coefficient of 0.9932) with the average copy number obtained by digitalPCR of cDNA prepared from bulk lysates. FIG. 20D shows 2072 single cellmeasurements of the expression of 9 miRNA in K562 cells. Reflectedhistograms represent the expression distributions for each miRNA.

Single cell measurements revealed distinct patterns of miRNA expression,with miR-16, miR-92, and miR-17-5p each exhibiting unimodal and tightlyregulated distributions, while miR-223, miR-196a, and miR-145 showedmulti-modal distributions and a high level of cellular heterogeneity.For the lowest abundance miRNA, miR-200a, expression was detected inonly a small fraction of cells and at levels below ˜5 copies per cell.The average miR-200a copy number over all cells was within factor of twoof that obtained by digital PCR (0.2 copies per cell). In contrast,miR-92 was found to be the most abundant miRNA and was present atapproximately 60,000 copies per cell. These measurements establishedmiRNA quantification in single cells with a dynamic range of greaterthan 104 and at single molecule sensitivity.

To illustrate the utility of single cell measurements in preciselyassessing differences in both the average expression and theheterogeneity between two different cell populations, the expressionlevels of miR-16 and miR-223 in K562 cells were compared to those inCA1S cells, a human embryonic stem cell line (hESC). Although miR-16 wasfound to be expressed in hESC at similar levels to K562 (ΔCT=0.6),approximately a two-fold greater variability in expression was observed(mean CT=22.0, s.d.=0.7) (FIG. 20A). In contrast, when compared to K562,single CA1S cell measurements of miR-223 showed strong down-regulation,with miR-223 detected in only 3.6% of cells.

Example 2.6 Co-Regulation of miR-145 and OCT4 in Single Cells

The measurement of multiple transcripts in single cells allows forquantitative measurements of gene co-regulation that would otherwise bemasked by cellular heterogeneity. To demonstrate this capability, anoptically multiplexed single cell RT-qPCR assay was conducted to studythe co-regulation of miR-145 and OCT4, a known target of miR-145, duringthe differentiation of hESCs (FIG. 21A-C). FIG. 21A is a scatterplot ofa multiplexed analysis of the co-expression of OCT4 and miR145 indifferentiating hESC. Points are color-coded to represent single cellmeasurements (N=547) for each time point. Crosses represent populationmean copy number. FIGS. 21B and 21C are histograms showing thedistribution of each transcript are projected on the axes with the meancopy number indicated by a dashed line. A total of 1094 single cellmeasurements were performed at 0, 4, 6, and 8 days of differentiation.Cell distributions at each time point were used to map out the evolutionof these transcripts and showed that average miR-145 levels increasedapproximately 20 fold (copy numbers: D0: mean=18.9, s.d.=25.5, D8:mean=380.3, s.d.=259.4) over 8 days. Increases in miR-145 wereaccompanied by progressive down-regulation of OCT4, ultimately reachingan average of 30-fold suppression (copy numbers: D0: mean=755.7,s.d.=306.4, D8: mean=27.8, s.d.=124.5) after 8 day. This wasindependently verified by mRNA-FISH, as shown in FIG. 22 images ofmRNA-FISH of OCT4 counterstained with DAPI in CA1S cells. FIG. 22A is arepresentative image of mRNA-FISH of OCT4 in a CA1S cell after 7 days ofFBS differentiation. The estimate of average copy number of OCT4 mRNA asdetermined by manual inspection of image stacks is 42 (s.d.=41, N=6).FIG. 22B is a representative image of undifferentiated CA1S cells. Theestimate of average copy number as determined by manual inspection ofimage stacks is 988 (s.d.=368, N=6). The scale bar=10 am.

Notably, single cell analysis at day 6 showed a bimodal distribution inboth OCT4 and miR-145, revealing a transition of cellular state. Withoutwishing to be bound by hypothesis, it is believed that this likelyreflects the spontaneous differentiation of a subpopulation of cells.The observed single cell dynamics of miR-145 and OCT4 co-regulation arenot apparent in population measurements, highlighting the use ofscalable single cell transcriptional analysis in correlating molecularsignatures to cellular decision making.

Example 2.7 SNV Detection in Primary Cells

To establish the specificity of the method, multiplexed measurements ofmRNA single nucleotide variants (SNV) were used to assess the genomicheterogeneity within a primary tumor sample. A total of 117 single cellsisolated from a plural effusion of a metastatic breast cancer wereassayed for the expression of a SNV mutant of the transcription factorSP1, previously identified by deep sequencing (73) (FIG. 21D). Primerswere designed using sequences flanking the SNV location and do notdiscriminate between the genomic DNA and mRNA transcript. Of the 117primary cells analyzed, 22 (18.8%) were heterozygous for the mutant andwildtype allele, 85 (72.6%) were homozygous wildtype, 1 (0.9%) washomozygous mutant and the transcripts were undetected in 9 (7.7%). TheSP1 mutation was not detected in 37 control K562 cells, and thewild-type transcript was not detected in only 2 of these cells. In theabsence of copy number alterations in the primary sample, these observedfrequencies would suggest a mutant to wild-type SP1 ratio of 11.2%(18.8×1+0.9×2=20.6 mutant to 18.8×1+72.6×2=164 wild-type). However,using digital PCR on purified DNA from the primary sample, the ratio ofmutant to wild-type SP1 alleles was found to be 18.7±2.3%, in agreementwith the previously reported ratio of 21.9%, obtained by deep sequencing(73). Given that the frequency of tumor cells within the original samplewas approximately 89% (73), both DNA molecule counting and single cellRNA expression measurements show that the metastasis of this tumor isderived from multiple cancer cell lineages.

Example 3 Library Construction

A K52 cell suspension was loaded directly into a microfluidic device aspreviously described. Captured cells immobilized in the traps werewashed with PBS solution for 10 min at 3 psi. Chambers containing singlecells were isolated by actuation of valves, heat lysed (85 deg 5 min)and filled via a second inlet in the cell capture chamber upstream ofthe cell trap, with a first reaction mix comprising 3′ miRNA adaptersfor ligation.

Next, a ligation mix that ligates 5′ DNA adapters to the 5′ end ofmiRNAs was added through the second inlet. An interface valve betweenthe cell capture chamber, containing the lysed cell and the firstreaction mix, and a larger first auxiliary chamber was opened, allowingthe contents of the cell capture chamber to be flushed into the firstauxiliary chamber along with the ligation mix under 5 psi of pressure.

Following ligation in the first auxiliary chamber, a cDNA synthesis mixwas added via the second inlet to synthesize cDNA from the 5′adapter-miRNA-3′ adapter molecules. An interface valve between the firstauxiliary chamber, containing the lysed cell and the first reaction mixand ligation mix) and a larger second auxiliary chamber was opened toallow the contents of the second auxiliary chamber to be flushed intothe second auxiliary chamber with the cDNA synthesis mix under 5 psi ofpressure. Following cDNA synthesis, the reaction products were elutedthrough an elution port with 5 uL of fresh PCR mix for cDNAamplification, and subsequent high throughput sequencing on an Illuminasequencing instrument.

Sequencing identified a number of RNA species (FIG. 23), including ahigh percentage of miRNAs. The miRNA species found in one single cell islisted in Table. In total, 11 single cells were processed in a singlemicrofluidic device along with positive (purified RNA) and a negative(no cell) controls.

Example 4

A microfluidic chip consisting of two fluidic paths was designed forsequencing and characterizing individual transcriptomes (FIG. 24). Thefirst path leads from an inlet port, through a horizontal channelcontaining eight hydrodynamic cell traps, to an outlet. The horizontalchannel can be partitioned to isolate the eight traps into eightindividual cell capture chambers and one, equivalent volume, emptychamber for use as a control. The second fluidic path leads from second“reagent” inlets at the top of the chip and flows vertically down thechip into a series of nine reaction columns. Each columns intersects apartitioned cell capture chambers, or the control volume, and then flowsthrough a sieve valve to an individual outlet port. A sieve valve is a“leaky” valve, allowing fluid to flow through, but stopping largerobjects, such as RNA capture beads.

This chip is able to perform single-cell capture, lysis, mRNApurification and reverse-transcription. Briefly, mRNA capture beads areloaded through the reagent inlets and stacked against the sieve valves.A set of valves is then closed to isolate and protect the stackedcolumns. Next, cells are introduced through the horizontal channel andflowed until each cell trap has been filled, after which time the cellscan be washed with clean buffer run through the horizontal channel. Thecell capture chambers are then partitioned to isolate the cells, and thechip is transferred to flat-bed thermocycler for a heat lysis. Thevertical path is then opened, and the lysate is passed over the beadcolumns, capturing the mRNA. As the lysate passes from the cell capturechamber to the column, it flows through a lysate dilution chamber. Thischamber is important as pure lysate contains proteins and other materialwhich can aggregate and clog the bead column. This dilution steps servesto avoid this. Once capture is complete, the chip is again incubated ona thermocycler, and reverse transcription mix introduced through thereaction inlet and flowed over the column. Finally, the sieve valve isopened, and beads, with cDNA now bound to them, are flowed to theirrespective outlet ports. The cDNA can then be used for PCR amplificationand subsequent sequencing. In addition, random sequences can beincorporated on the capture beads, resulting in each captured mRNAmolecule being given a unique barcode. As a result, the sequencing datacan be used as a digital count of transcript abundance.

Results from the characterization of four cells in this way arepresented in Tables 3 and 4.

Example 5

FIG. 25 shows a cell capture chamber with a downstream sieve valve.Functionalized beads (e.g. functionalized with oligo-dTs) are stackedagainst the sieve valves under flow pressure (FIG. 25B), and then asingle cell (or predetermined number of cells) is added to the chamberby deterministic cell loading, in which fluid routing through the use ofvalves can be used to isolate single cells from a bulk sample, or theuse of cell traps as described above (FIG. 25A). Cells may beimmobilized at each directed location by a hydrodynamic cell trap or bya leaky ‘sieve’ valve. By flowing a chemical lysis reagent through thechamber, the cell is lysed, and the substrate (e.g. mRNA) is capturedand purified on the beads. Subsequent reagents may be flowed through thereaction chamber while keeping the substrate-bound beads immobilized bythe sieve valve. The chamber may be sealed by (non-leaky) valves tocarry out each reaction step.

TABLE 3 Results from sequencing barcoded cDNA produced on-chip. Thismethod is intended to quantify transcript numbers rather than to obtainfull gene sequences Unique Transcripts Molecules 3rd Most AbundantMapped Sequenced Most Abundant Transcript 2nd Most Abundant TransciptTranscript Cell 1 1382 1909 acetyl-Coenzyme A 18S ribosomal RNA (x25)28S ribosomal RNA (x14) carboxylase beta (x49) Cell 2 2041 3205acetyl-Coenzyme A 18S ribosomal RNA (x35) multiple EGF-like-domains 8carboxylase beta (x75) (x30) Cell 3 1566 2330 18S ribosomal RNA (x114)28S ribosomal RNA (x50) acetyl-Coenzyme A carboxylase beta (x36) Cell 42984 5159 18S ribosomal RNA (x172) acetyl-Coenzyme A 28S ribosomal RNA(x57) carboxylase beta (x110) On-Chip 31 36 similar to oncomodulin (x3)keratin 24 (x3) acetyl-Coenzyme A Control carboxylase beta (x2) PCR 6273 zinc finger protein 182 (x12) Control

TABLE 4 Results from Illumina sequencing of single-cell, wholetranscriptomes (no randomers) using a microfluidic chip. Genes Genesdetected detected % at at Total Total Aligned % Dups Genes >=2 >=10Coverage Description reads Gbp reads Aligned total detected reads reads5′/3′ Single Cell 1 14341272 0.64 10336447 72.1 91.8 1603 1304 668 86.7Single Cell 2 14916262 0.66 9171788 61.5 74.7 2719 2443 1864 14.9 SingleCell 3 12994578 0.57 9154643 70.4 71.9 2197 1904 1271 42.9 No Cell14426522 0.64 9372123 65 94 1494 1185 582 43.4 Cont. 1 No Cell 161018060.71 10098883 62.7 94.5 6150 3283 528 15.9 Cont. 2

Example 6

FIG. 26 illustrates a microfluidic device for magnetic immobilization offunctionalized beads to facilitate multi-step reactions in a singlechamber. Cells are isolated in capture chambers adjacent to largerreaction chambers. Functionalized magnetic beads, e.g. with poly-Toligonucleotides, are suspended in lysis reagent and then injectedthrough cell capture chamber and into a reaction chamber, lysing thecell(s) in the process. After binding substrate (e.g. mRNA), the beadsare immobilized at the top of the chamber (or any suitable wall of thechamber) by a magnetic field, thereby allowing the fluidic contents ofthe reaction chamber to be replaced. This allows for substratepurification and multi-step processing of the bound substrate within asingle chamber.

Example 7

FIG. 27 is a schematic diagram of a microfluidic device integratingsingle cell processing with microarray spotting to address differentreagents (i.e. primers/probes) to different reactions according to oneembodiment of the invention. In this a captured cell is heat lysed, andthe cell lysate is then pushed with each subsequent reagent into theadjacent chambers (e.g., for reverse transcription (RT), and PCR). Thereaction products are recoverable through an outlet in the chamber. Inthis design a laser may be used to ablate fluidic vias (interconnects)down to chambers aligned over an array of spotted reagents. Using arobotic microarray spotter, these spots can be used to provide eachreaction with a different primer or probe. Primers may be spotted with aunique identifying sequence of DNA bases. This “barcode” is incorporatedin each single cell reaction and facilitates DNA and/or RNA transcriptsequencing of many single cells in parallel, with the cell of origin foreach sequence encoded in the barcode.

Example 8

A microfluidic device for integrated single cell digital PCR accordingto one embodiment of the invention is show in FIG. 28. This device camperform all fluid handling steps for parallel single moleculeamplification and counting of RNA transcripts from single cells. Thedevice combines the cell processing presented in Example 2 with an arrayof 2000×1 nL chambers for digital PCR. Cells are captured by prior tobeing washed and lysed. The contents are passed to adjacent, largerchambers for reverse transcription. Finally, the DNA template (RTproduct) is pushed into the array of digital PCR chambers with PCRreagent. To mix the PCR solution and template molecules, the digitalarray was designed as a large bifurcating rotary mixer where the fluidrevolves around the array by the use of a peristaltic pump (FIG. 29).After mixing, valves (in red) partition the lanes into 2000 reactionchambers. Digital PCR is performed, and the number of wells withamplification are counted to directly count single molecules fromsample, i.e. single cell. This strategy, when combined with high-densitydigital PCR arrays, enables the high-throughput analysis of single cellsby digital PCR with excellent efficiency in the transfer of cell-derivednucleic acids into the digital array; this is difficult to achieve ifcells are processed in tubes and then transferred to a device fordigital PCR analysis. It should be appreciated that this geometry may beused along with a chemical or enzymatic DNA fragmentation step to dodigital PCR analysis on genomic DNA and that this may find applicationsin the analysis of copy number variations from single cells.

Example 9

A microfluidic device for sequentially diluting cell products by passingthem to larger chambers according to one embodiment of the invention,that may or may not include active mixing, is shown in FIG. 30. Sevensequential chambers are shown. Reagents are pushed from one chamber tothe next by dilution with a subsequent reagent. Previous chambers canalso be bypassed or flushed. Active mixing is used to increase the speedof mixing in large chambers. The right panel is a fluorescence image ofactive mixing by using valves as peristaltic pump in channels aroundoutside of chamber to circulate solution. This design is optimized formultistep reactions such as described by Tang et al. (80) for theamplification of RNA from single cells to generate sufficient materialfor sequencing. For instance, multiple steps, including cell capture,cell lysis, cDNA synthesis, EXO1 degradation of primers, poly-A tailing,second strand synthesis, and PCR, can be executed by sequential transferof cell contents between chambers. It should be noted here that eachsubsequent chamber provides a 3:1 dilution in order to achieve efficienttransfer of contents at each step, and that this requires that theconcentration of reagents at each step be chosen appropriately. Further,it should be appreciated that different dilution ratios are possible andmay be desirable.

Example 10 Spatial Multiplexing

In yet another embodiment of the invention, cell lysate may be splitinto multiple chambers for parallel performance of different assays.FIG. 31 presents a microfluidic device for (low) special multiplexingaccording to one embodiment of the invention. Spatial multiplexingallows multiple assays to be spatially defined, and removes the opticallimitations The device features cell processing units as described inExample 2, wherein a single cell is hydrodynamically trapped, washed andlysed. The lysate (DNA, RNA, or template) is transferred to the adjacentchamber by pushing with reverse transcription (RT) reagent. After RT,the product (cDNA) may be sequentially transferred and processed tomultiple adjacent chambers. In FIG. 31 (below), a single larger chamberis illustrated that could be used for mixing the template and PCRsolution by diffusion, and/or for pre-amplification of the template byPCR.

For multiplexing the template is pushed (with PCR reagent) down achannel with multiple (e.g. 10) chambers. The channel loops back ontoitself, and template and reagents may be mixed by a rotary mixer. Aftermixing, the channel is partitioned into individual chambers. Differentassays (i.e. primers/probes) are loaded into adjacent chambers (fluidrouting facilitated by laser ablated inter-layer connections). The valveseparating the combined PCR/template solution and the primer/probeassays is opened and the contents are allowed to mix by diffusion (analternative design could combine these two solutions by advection). Inthis way, the contents of a single cell can be interrogated for multipletargets (i.e. different transcripts).

Example 11

In yet another embodiment of the invention, a microfluidic device couldbe used to treat single cells (or a pre-determined number of cells)prior to analysis. For example, immobilized cells can be stimulated inany number of ways (e.g. by chemicals, pathogens, electricity, etc.) andlysed, with lysates transferred to reaction chambers for analysis. Forexample, cells positioned in traps could be subjected to stimulation bysignaling molecules included during the wash step. Following a definedamounts of time, potentially varied for different cells, the cells maythen be lysed and the contents transferred to auxiliary chambers for theamplification and/or quantification of nucleic acids by RT-qPCR, digitalPCR, sequencing, microarray analysis, and so forth. This would be usefulfor studying the early transcriptional events that result fromstimulation with single cell precision, temporal resolution andthroughput that is not possible using alternative methods.

A person skilled in the art will further understand that the method neednot be destructive. For example, immobilized cells can be stimulated,and subsequent cell secretions could be washed to downstream reactionchambers for analysis. The intact cells could then be subjected tofurther treatment or recovered for culturing. Such secretions couldinclude metabolites, hormones, cytokines, other proteins, miRNA, mRNA,and so forth. As an example of this the secreted proteins from a cellcould be quantified using a fluorescent sandwich assay in whichantibodies are used to capture and detect the protein of interest on asolid surface such as the channel walls or an immobilized microbead.Alternatively, secreted proteins could be detected using immuno PCRapproaches with qPCR performed in a downstream chamber. Alternativelydigital protein counting could be performed using enzymaticamplification and detection through cleavage of a fluorescent substrateor by performing digital immuno PCR wherein fewer than one protein ispresent in each of an array of downstream reactors. It should beappreciated by those skilled in the art that there are many possibleassays that can be performed on DNA, RNA, proteins, and metabolitessecreted from single cells.

Example 12

In another embodiment of the invention, microfluidic devices can be usedto isolate cells for culture analysis under flow. In particular, if thetraps are highly efficient and can only contain a single cell, then anarray of traps in a channel could be used to sequentially catch theprogeny of a cell (thereby allowing for lineage analysis). Suchembodiments are of utility in the study of asymmetric division of stemcells where the progeny of a cell may be programmed to a differentiationstate that is not identical to the cell of origin. For instance, thepositioning of a single cell at the start of an array of traps could beused, through cell division, to populate the remaining traps in thearray. The cells could then be individually processed to amplify andquantify mRNA or miRNA expression and thereby obtain informationregarding the transcriptional state of stem cells and their progenyunder defined conditions.

Example 13

In yet another embodiment of the invention, a microfluidic device couldbe used to trap and immobilize cells to improve transfection. In such anembodiment rare cell types could be immobilized and exposed to the flowof a solution of viruses to increase the efficiency of cell-virusinteraction. In one scenario the cells could be positioned downstream ofother cells that produce the virus of interest.

Example 14

In yet another embodiment of the invention, cell traps may be used tofix and stain cells, either pre- or post-lysis, including antibodystaining, mRNA FISH, FISH, or chemical staining.

Example 15

In yet another embodiment of the invention, the cell corpse may beretained within a trap after lysis while cell contents are passed todownstream chambers, thereby enabling process of both DNA and othercontents (e.g. RNA or protein) separately. For instance, chemical lysiswith the detergent NP40 is known to leave the nucleus of cells intact.Alternatively, heat lysis may be used to disrupt the membrane, releasingcytosolic components, while preserving the localization of the genomicDNA.

Operation

While specific embodiments of the invention have been described andillustrated, such embodiments should be considered illustrative of theinvention only and not as limiting the invention as construed inaccordance with the accompanying claims.

REFERENCES

-   1. Taniguchi K, Kajiyama T, & Kambara H (2009) Quantitative analysis    of gene expression in a single cell by qPCR. Nat Methods    6(7):503-506.-   2. Warren L, Bryder D, Weissman I L, & Quake SR (2006) Transcription    factor profiling in individual hematopoietic progenitors by digital    RT-PCR. Proc Natl Acad Sci USA 103(47):17807-17812.-   3. Raj A, van den Bogaard P, Rifkin S A, van Oudenaarden A, & Tyagi    S (2008) Imaging individual mRNA molecules using multiple singly    labeled probes. Nature Methods 5(10):877-879.-   4. Larsson C, Grundberg I, Soderberg O, & Nilsson M (2010) In situ    detection and genotyping of individual mRNA molecules. Nature    Methods 7(5):395-U381.-   5. Tang F, et al. (2009) mRNA-Seq whole-transcriptome analysis of a    single cell. Nat Methods 6(5):377-382.-   6. Bengtsson M, Hemberg M, Rorsman P, & Stahlberg A (2008)    Quantification of mRNA in single cells and modelling of RT-qPCR    induced noise. BMC Mol Biol 9:63.-   7. Zare R N & Kim S (2010) Microfluidic platforms for single-cell    analysis. Annu Rev Biomed Eng 12:187-201.-   8. Sims C E & Allbritton N L (2007) Analysis of single mammalian    cells on-chip. Lab Chip 7(4):423-440.-   9. Wheeler A R, et al. (2003) Microfluidic device for single-cell    analysis. Anal Chem 75(14):3581-3586.-   10. Skelley A M, Kirak O, Suh H, Jaenisch R, & Voldman J (2009)    Microfluidic control of cell pairing and fusion. Nat Methods    6(2):147-152.-   11. Marcus J S, Anderson W F, & Quake SR (2006) Microfluidic    single-cell mRNA isolation and analysis. Anal Chem 78(9):3084-3089.-   12. Bontoux N, et al. (2008) Integrating whole transcriptome assays    on a lab-on-a-chip for single cell gene profiling. Lab on a Chip    8(3):443-450.-   13. Zhong J F, et al. (2008) A microfluidic processor for gene    expression profiling of single human embryonic stem cells. Lab Chip    8(1):68-74.-   14. Guo G J, et al. (2010) Resolution of Cell Fate Decisions    Revealed by Single-Cell Gene Expression Analysis from Zygote to    Blastocyst. Developmental Cell 18(4):675-685.-   15. Petriv O I, et al. (2010) Comprehensive microRNA expression    profiling of the hematopoietic hierarchy. Proc Natl Acad Sci USA    107(35):15443-15448.-   16. Diehn M, et al. (2009) Association of reactive oxygen species    levels and radioresistance in cancer stem cells. Nature    458(7239):780-783.-   17. Toriello N M, et al. (2008) Integrated microfluidic bioprocessor    for single-cell gene expression analysis. Proc Natl Acad Sci USA    105(51):20173-20178.-   18 Rieseberg, M., Kasper, C., Reardon, K. & T, S. Flow cytometry in    biotechnology. Applied Microbiology and Biotechnology 56, 350-360    (2001).-   19 Cohen, D. et al. Chemical Cytometry: Fluorescence-Based    Single-Cell Analysis. Annual Review of Analytical Chemistry 1,    165-190, doi:doi:10.1146/annurev.anchem. 1.031207.113104 (2008).-   20 Veening, J.-W., Smits, W. K., Hamoen, L. W. & Kuipers, O. P.    Single cell analysis of gene expression patterns of competence    development and initiation of sporulation in <i>Bacillus    subtilis</i> grown on chemically defined media. Journal of Applied    Microbiology 101, 531-541 (2006).-   21 Kato, Y. et al. Real-time functional imaging for monitoring    miR-133 during myogenic differentiation. The International Journal    of Biochemistry & Cell Biology 41, 2225-2231, doi:DOI:    10.1016/j.biocel.2009.04.018 (2009).-   22 Zhang, L. et al. Whole genome amplification from a single cell:    implications for genetic analysis. Proc. Natl. Acad. Sci. U.S.A. 89,    5847-5851 (1992).-   23 Emmert-Buck, M. R. et al. Laser Capture Microdissection. SCIENCE    274, 998-1001, doi:10.1126/science.274.5289.998 (1996).-   24 Orba, Y. et al. Application of laser capture microdissection to    cytologic specimens for the detection of immunoglobulin heavy chain    gene rearrangement in patients with malignant lymphoma. Cancer    Cytopathology 99, 198-204 (2003).-   25 Stich, M. et al. Live Cell Catapulting and Recultivation.    Pathology—Research and Practice 199, 405-409, doi:Doi:    10.1078/0344-0338-00437 (2003).-   26 Li, P. C. H., Camprieu, L. d., Cai, J. & Sangar, M. Transport,    retention and fluorescent measurement of single biological cells    studied in microfluidic chips. Lab on a Chip 4, 174-180 (2004).-   27 Li, X. & Li, P. C. H. Microfluidic Selection and Retention of a    Single Cardiac Myocyte, On-Chip Dye Loading, Cell Contraction by    Chemical Stimulation, and Quantitative Fluorescent Analysis of    Intracellular Calcium. Anal. Chem. 77, 4315-4322,    doi:10.1021/ac048240a (2005).-   28 Di Carlo, D., Aghdam, N. & Lee, L. P. Single-Cell Enzyme    Concentrations, Kinetics, and Inhibition Analysis Using High-Density    Hydrodynamic Cell Isolation Arrays. Anal. Chem. 78, 4925-4930,    doi:10.1021/ac060541s (2006).-   29 Rowat, A. C., Bird, J. C., Agresti, J. J., Rando, O. J. &    Weitz, D. A. Tracking lineages of single cells in lines using a    microfluidic device. Proceedings of the National Academy of Sciences    106, 18149-18154, doi:10.1073/pnas.0903163106 (2009).-   30 Kobel, S., Valero, A., Latt, J., Renaud, P. & Lutolf, M.    Optimization of microfluidic single cell trapping for long-term    on-chip culture. Lab on a Chip 10, 857-863 (2010).-   31 King, K. R. et al. A high-throughput microfluidic real-time gene    expression living cell array. Lab on a Chip 7, 77-85 (2007).-   32 Marcy, Y. et al. Dissecting biological “dark matter” with    single-cell genetic analysis of rare and uncultivated TM7 microbes    from the human mouth. Proc. Natl. Acad. Sci. U.S.A. 104, 11889-11894    (2007).-   33 Voldman, J., Gray, M. L., Toner, M. & Schmidt, M. A. A    microfabrication-based dynamic array cytometer. Anal. Chem. 74,    3984-3990, doi:10.1021/ac0256235 (2002).-   34 Braschler, T., Johann, R., Heule, M., Metref, L. & Renaud, P.    Gentle cell trapping and release on a microfluidic chip by in situ    alginate hydrogel formation. Lab on a Chip 5, 553-559 (2005).-   35 Neuman, K. C., Chadd, E. H., Liou, G. F., Bergman, K. &    Block, S. M. Characterization of photodamage to Escherichia coli in    optical traps. Biophys. J. 77, 2856-2863 (1999).-   36 Bartel, D. P. MicroRNAs: Genomics, Biogenesis, Mechanism, and    Function. Cell 116, 281-297, doi:Doi: 10.1016/s0092-8674(04)00045-5    (2004).-   37 O'Donnell, K. A., Wentzel, E. A., Zeller, K. I., Dang, C. V. &    Mendell, J. T. c-Myc-regulated microRNAs modulate E2F1 expression.    Nature 435, 839-843,    doi:http://www.nature.com/nature/journal/v435/n7043/suppinfo/nature03677_S1.html    (2005).-   38 Chen, C.-Z., Li, L., Lodish, H. F. & Bartel, D. P. MicroRNAs    Modulate Hematopoietic Lineage Differentiation. SCIENCE 303, 83-86,    doi:10.1126/science.1091903 (2004).-   39 Miska, E. et al. Microarray analysis of microRNA expression in    the developing mammalian brain. Genome Biology 5, R68 (2004).-   40 Sindelka, R., Jonak, J., Hands, R., Bustin, S. A. & Kubista, M.    Intracellular expression profiles measured by real-time PCR    tomography in the Xenopus laevis oocyte. Nucleic Acids Research 36,    387-392 (2008).-   41 Chang, S., Johnston, R. J. & Hobert, O. A transcriptional    regulatory cascade that controls left/right asymmetry in    chemosensory neurons of C. elegans. Genes & Development 17,    2123-2137, doi:10.1101/gad.1117903 (2003).-   42 Krichevsky, A. M., King, K. S., Donahue, C. P., Khrapko, K. &    Kosik, K. S. A microRNA array reveals extensive regulation of    microRNAs during brain development. RNA 9, 1274-1281,    doi:10.1261/rna.5980303 (2003).-   43 Niu, Q.-W. et al. Expression of artificial microRNAs in    transgenic Arabidopsis thaliana confers virus resistance. Nat    Biotech 24, 1420-1428,    doi:http://www.nature.com/nbt/journal/v24/n11/suppinfo/nbt1255_S1.html    (2006).-   44 Scaria, V., Hariharan, M., Maiti, S., Pillai, B. &    Brahmachari, S. Host-virus interaction: a new role for microRNAs.    Retrovirology 3, 68 (2006).-   45 Calin, G. A. et al. MicroRNA profiling reveals distinct    signatures in B cell chronic lymphocytic leukemias. Proc. Natl.    Acad. Sci. U.S.A. 101, 11755-11760 (2004).-   46 Cui, J.-W. et al. Retroviral insertional activation of the Fli-3    locus in erythroleukemias encoding a cluster of microRNAs that    convert Epo-induced differentiation to proliferation. Blood 110,    2631-2640, doi:10.1182/blood-2006-10-053850 (2007).-   47 Lu, J. et al. MicroRNA expression profiles classify human    cancers. Nature 435, 834-838,    doi:http://www.nature.com/nature/journal/v435/n7043/suppinfo/nature03702_S1.html    (2005).-   48 Ikeda, S. et al. Altered microRNA expression in human heart    disease. Physiol. Genomics 31, 367-373,    doi:10.1152/physiolgenomics.00144.2007 (2007).-   49 Thum, T., Catalucci, D. & Bauersachs, J. MicroRNAs: novel    regulators in cardiac development and disease. Cardiovasc Res 79,    562-570, doi:10.1093/cvr/cvn137 (2008).-   50 Thum, T. et al. MicroRNAs in the Human Heart: A Clue to Fetal    Gene Reprogramming in Heart Failure. Circulation 116, 258-267,    doi:10.1161/circulationaha.107.687947 (2007).-   51 Wang, N., Zhou, Z., Liao, X. & Zhang, T. Role of microRNAs in    cardiac hypertrophy and heart failure. IUBMB Life 61, 566-571    (2009).-   52 Gibson, U. E., Heid, C. A. & Williams, P. M. A novel method for    real time quantitative RT-PCR. Genome Research 6, 995-1001,    doi:10.1101/gr.6.10.995 (1996).-   53 Ginzinger, D. G. Gene quantification using real-time quantitative    PCR: An emerging technology hits the mainstream. Experimental    Hematology 30, 503-512, doi: Doi: 10.1016/s0301-472x(02)00806-8    (2002).-   54 Nolan, T., Hands, R. E. & Bustin, S. A. Quantification of mRNA    using real-time RT-PCR. Nat. Protocols 1, 1559-15382 (2006).-   55 Bengtsson, M., Hemberg, M., Rorsman, P. & Stahlberg, A.    Quantification of mRNA in single cells and modelling of RT-qPCR    induced noise. Bmc Molecular Biology 9 (2008).-   56 Taniguchi, K., Kajiyama, T. & Kambara, H. Quantitative analysis    of gene expression in a single cell by qPCR. Nature Methods 6,    503-U550 (2009).-   57 Tang, F. C., Hajkova, P., Barton, S. C., Lao, K. Q. &    Surani, M. A. MicroRNA expression profiling of single whole    embryonic stem cells. Nucleic Acids Research 34 (2006).-   58 Tang, F. C. et al. 220-plex microRNA expression profile of a    single cell. Nature Protocols 1, 1154-1159 (2006).-   59 Kamme, F. et al. Single-Cell Microarray Analysis in Hippocampus    CA1: Demonstration and Validation of Cellular Heterogeneity. J.    Neurosci. 23, 3607-3615 (2003).-   60 Kloosterman, W. P., Wienholds, E., de Bruijn, E., Kauppinen, S. &    Plasterk, R. H. A. In situ detection of miRNAs in animal embryos    using LNA-modified oligonucleotide probes. Nat Meth 3, 27-29,    doi:http://www.nature.com/nmeth/journal/v3/n1/suppinfo/nmeth843_S1.html    (2006).-   61 Lu, J. & Tsourkas, A. Imaging individual microRNAs in single    mammalian cells in situ. Nucleic Acids Research 37 (2009).-   62 Neely, L. A. et al. A single-molecule method for the quantitation    of microRNA gene expression. Nat Meth 3, 41-46,-   63 Unger, M. A. et al. Microfabricated Elastomeric Valve and Pump    Systems USA patent (2005).-   64 Lee, R. C., Feinbaum, R. L. & Ambros, V. The C. elegans    heterochronic gene lin-4 encodes small RNAs with antisense    complementarity to lin-14. Cell 75, 843-854, doi:Doi:    10.1016/0092-8674(93)90529-y (1993).-   65 Nelson, P. T. et al. Microarray-based, high-throughput gene    expression profiling of microRNAs. Nat Meth 1, 155-161,    doi:http://www.nature.com/nmeth/journal/v1/n2/suppinfo/nmeth717_S1.html    (2004).-   66 Thomson, J. M., Parker, J., Perou, C. M. & Hammond, S. M. A    custom microarray platform for analysis of microRNA gene expression.    Nat Meth 1, 47-53,    doi:http://www.nature.com/nmeth/journal/v1/n1/suppinfo/nmeth704_S1.html    (2004).-   67 Willenbrock, H. et al. Quantitative miRNA expression analysis:    comparing microarrays with next-generation sequencing. RNA 15,    2028-2034, doi:doi: 10.1261/ma.1699809 (2009).-   68 Chen, C. et al. Real-time quantification of microRNAs by    stem-loop RT-PCR. Nucl. Acids Res. 33, e179-, doi:10.1093/nar/gni178    (2005).-   69 Livak, K. J., Flood, S. J., Marmaro, J., Giusti, W. & Deetz, K.    Oligonucleotides with fluorescent dyes at opposite ends provide a    quenched probe system useful for detecting PCR product and nucleic    acid hybridization. Genome Research 4, 357-362 (1995).-   70 Unger, M. A., Chou, H. P., Thorsen, T., Scherer, A. &    Quake, S. R. Monolithic microfabricated valves and pumps by    multilayer soft lithography. SCIENCE 288, 113-116 (2000).-   71 Thorsen T, Maerkl S J, & Quake S R (2002) Microfluidic    large-scale integration. Science 298(5593):580-584.-   72 Warren L, Bryder D, Weissman I L, & Quake S R (2006)    Transcription factor profiling in individual hematopoietic    progenitors by digital RT-PCR. Proc Natl Acad Sci USA    103(47):17807-17812.-   73 Shah S P, et al. (2009) Mutational evolution in a lobular breast    tumour profiled at single nucleotide resolution. Nature    461(7265):809-813.-   74 Schulze H G, et al. (2010) Assessing Differentiation Status of    Human Embryonic Stem Cells Noninvasively Using Raman    Microspectroscopy. Analytical Chemistry 82(12):5020-5027.-   75 Adewumi O, et al. (2007) Characterization of human embryonic stem    cell lines by the International Stem Cell Initiative. Nature    Biotechnology 25(7):803-816.-   76 Ludwig T E, et al. (2006) Feeder-independent culture of human    embryonic stem cells. Nat Methods 3(8):637-646.-   77. Lozzio B B, Lozzio C B, Bamberger E G, & Feliu A S (1981) A    multipotential leukemia cell line (K-562) of human origin. Proc Soc    Exp Biol Med 166(4):546-550.-   78. Yuan J Y, et al. (2009) MicroRNA-223 reversibly regulates    erythroid and megakaryocytic differentiation of K562 cells. J Cell    Mol Med 13(11-12):4551-4559.-   79. Landgraf P, et al. (2007) A mammalian microRNA expression atlas    based on small RNA library sequencing. Cell 129(7):1401-1414.-   80. Tang F C, Barbacioru C, Nordman E, Li B, Xu N L, Bashkirov V I,    Lao K Q, Surani M A: RNA-Seq analysis to capture the transcriptome    landscape of a single cell. Nature Protocols 5:516-535.

1. A microfluidic device comprising: (a) a cell capture chamber, havingat least one inlet and at least one outlet, wherein each inlet andoutlet has an open and a closed position, whereby when the inlet is inthe open position fluid is able to flow into the cell capture chamberand when the outlet is in the open position fluid is able to flow out ofthe cell capture chamber, and whereby when the inlet is in the closedposition fluid is prevented from flowing into the cell capture chamberand when the outlet is in the closed position fluid is prevented fromflowing out of the cell capture chamber, and wherein the direction offlow through the cell capture chamber dictates an upstream and adownstream orientation of the cell capture chamber; (b) a cell funnelpositioned within the cell capture chamber and operable to direct a cellpassing through the cell capture chamber towards one or more desiredlocations within the cell capture chamber; and (c) a cell trap,positioned generally downstream from the cell funnel, whereby the celltrap is positioned to receive a cell flowing downstream from the cellfunnel.
 2. The microfluidic device of claim 1, further comprising: (a) asecond inlet in fluid communication with a fluid injection channel, theupstream inlet having an open and a closed position, wherein the openposition allows for fluid to enter the cell capture chamber from thefluid injection channel and in the closed position prevents fluid flowinto the cell capture chamber from the fluid injection channel; and (b)a second outlet in fluid communication with one or more auxiliarychambers, the downstream outlet having an open and a closed position,wherein the open position allows for fluid to exit the cell capturechamber and enter the one or more auxiliary chambers and in the closedposition prevents fluid flow into the one or more auxiliary chambersfrom the cell capture chamber.
 3. The microfluidic device of claim 1,further comprising a second inlet in fluid communication with a fluidinjection channel, the second inlet having an open and a closedposition, wherein the open position allows for fluid to enter the cellcapture chamber from the fluid injection channel and in the closedposition prevents fluid flow into the cell capture chamber from thefluid injection channel, and wherein the volume of the cell capturechamber is expandable.
 4. The microfluidic device of claim 1, whereinthe cell capture chamber has one inlet and one outlet, wherein the inletis in fluid communication with a fluid injection channel, and whereinthe volume of the cell capture chamber is expandable.
 5. Themicrofluidic device of claim 1 wherein the cell funnel exerts a force todirect cells towards the one or more desired locations within the cellcapture chamber.
 6. The microfluidic device of claim 5, wherein theforce exerted on the cells by the cell funnel is selected from one ormore of the following: a mechanical force; a gravitational force; anelectromagnetic force; an electrostatic force; a magnetic force; anacoustic force; a hydrodynamic force; and an optical force.
 7. Themicrofluidic device of claim 5, wherein the force exerted on the cellsby the cell funnel is a hydrodynamic force. 8.-9. (canceled)
 10. Themicrofluidic device of claim 2, wherein the second outlet is in fluidcommunication with a first auxiliary chamber.
 11. The microfluidicdevice of claim 10, wherein the first auxiliary chamber is in fluidcommunication with a second auxiliary chamber, wherein there is a valvebetween the first and second auxiliary chambers, wherein the valve hasan open position to allow fluid flow from the first auxiliary chamber tothe second auxiliary chamber and a closed position to prevent fluid flowfrom the first auxiliary chamber to the second auxiliary chamber. 12.The microfluidic device of claim 10, wherein the first auxiliary chamberis in fluid communication with a second auxiliary chamber and the secondauxiliary chamber is in fluid communication with a third auxiliarychamber, wherein there is a valve between the first and second auxiliarychambers, wherein the valve has an open position to allow fluid flowfrom the first auxiliary chamber to the second auxiliary chamber and aclosed position to prevent fluid flow from the first auxiliary chamberto the second auxiliary chamber, wherein there is a valve between thesecond and third auxiliary chambers, wherein the valve has an openposition to allow fluid flow from the second auxiliary chamber to thethird auxiliary chamber and a closed position to prevent fluid flow fromthe second auxiliary chamber to the third auxiliary chamber.
 13. Themicrofluidic device of claim 2 wherein the volume of the auxiliarychambers is expandable. 14.-17. (canceled)
 18. The microfluidic deviceof claim 1 wherein the ratio between the second auxiliary chamber andthe first auxiliary chamber is 5:1.
 19. The microfluidic device of claim1 wherein the ratio between the expanded cell capture chamber and theunexpanded cell capture chamber is 5:1 or the ratio between the expandedfirst auxiliary chamber unexpanded first auxiliary chamber is 5:1. 20.(canceled)
 21. The microfluidic device of claim 1 wherein the cellfunnel comprises a pair of cell deflectors each having a proximal and adistal end, wherein the proximal ends are positioned at opposite sidesof the capture chamber, and wherein each distal end of the celldeflector is angled on the diagonal in a downstream direction relativeto the proximal ends, whereby the distal ends of the cell deflectorsprovide an opening sized to permit the passage of a cell between thedistal ends of the cell deflectors.
 22. (canceled)
 23. The microfluidicdevice of claim 1 wherein the cell trap provides for fluid flow throughthe cell trap.
 24. A cell capture and preparation method, the methodcomprising: (a) flowing cells in a fluid through a chamber; (b)funneling the cells in the fluid towards a cell trap; (c) capturing apredefined number of the cells within the chamber; (d) interrupting theflow of cells in the fluid; (e) washing the captured cells by flowing awash solution through the chamber, wherein the flow of wash solutionremoves contaminants from the chamber; and (f) sealing the predefinednumber of cells in the chamber.
 25. A cell capture and preparationmethod, the method comprising: (a) flowing cells in a fluid through achamber; (b) funneling the cells in the fluid towards a cell trap; (c)capturing a predefined number of the cells within the chamber; (d)interrupting the flow of cells in the fluid; (e) sealing the predefinednumber of cells in the chamber; and (f) washing the captured cells byflowing a wash solution through the chamber, wherein the flow of washsolution removes contaminants from the chamber. 26.-28. (canceled)
 29. Acell assay method, the method comprising: (a) directing cells to a cellcapture chamber, wherein the chamber has a volume of between 0.1 nL to100 nL; (b) capturing a predefined number of the cells within thechamber; (c) washing the captured cells by flowing a wash solutionthrough the chamber, wherein the flow of wash solution removescontaminants from the chamber; and (d) isolating the predefined numberof cells in the chamber. 30.-32. (canceled)
 33. A cell assay method, themethod comprising: (a) directing cells to a cell capture chamber,wherein the chamber has a volume of between 0.1 nL to 100 nL; (b)capturing a predefined number of the cells within the chamber; (c)washing the captured cells by flowing a wash solution through thechamber, wherein the flow of wash solution removes contaminants from thechamber; (d) isolating the predefined number of cells in the chamber;(e) lysing the cells within the chamber; (f) reverse transcribing theRNA released by the cell lysis; and (g) amplifying the cDNAs transcribedin (f) by polymerase chain reaction (PCR). 34.-36. (canceled)